Methods for diagnosing episodic movement disorders and related conditions

ABSTRACT

The present invention provides compositions and methods for research, diagnostic, drug screening, and therapeutic applications related to paroxysmal dystonic choreoathetosis and related conditions. In particular, the present invention provides mutations in the myofibrillogenesis regulator 1 (MR-1) gene associated with such conditions.

This application claims priority to U.S. Provisional Application Ser.No. 60/583,058, filed Jun. 25, 2004, herein incorporated by reference inits entirety.

The present invention was funded in part under grant funds from NationalInstitutes of Health Grant No. 1R01 NS045163-01. The government may havecertain rights in the invention.

FIELD OF THE INVENTION

The present invention provides compositions and methods for research,diagnostic, drug screening, and therapeutic applications related toparoxysmal dystonic choreoathetosis and related conditions. Inparticular, the present invention provides mutations in themyofibrillogenesis regulator 1 (MR-1) gene associated with suchconditions.

BACKGROUND OF THE INVENTION

Episodic phenomena are common in humans. These include (but are notlimited to) seizures, headaches, cardiac arrhythmias, episodic movementdisorders, and periodic paralyses. These disorders have strong geneticdeterminants and often affect people who are completely normal betweenattacks. Although episodic disorders of the brain, heart, and muscleseem quite different on the surface, they share many similarities. Theyoften surface in childhood or adolescence and frequently improve withaging. In addition to being episodic, attacks in all of these disorderscan often be precipitated by stress, fatigue, alcohol, and some dietaryfactors. The medications used to treat these disorders overlapsignificantly. Thus, insights gained by study of any of these disorderscan impact an understanding of other related disorders.

Paroxysmal Dystonic Choreoathetosis (Mendelian Inheritance in Man No.11880; hereinafter, “PDC”), also known as paroxysmal nonkinesigenicdyskinesia, is an episodic movement disorder in which attacks ofdystonia, chorea, and athetosis begin in childhood through earlyadulthood; involve the extremities, trunk, and face; and may causedysarthria or dysphagia. These episodes last from several minutes tomore than an hour and may occur several times each day (see, e.g.,Mount, L. A. and Reback S., Arch Neurol Psychiatry. 1940, 44:841-847;Demirkiran M. and Jankovic J., Ann Neurol. 1995, 38:571-579; Richards R.N. and Barnett, H. J. M. Neurology 1968, 18:461-469; Fahn S. J NeurolNeurosurgPsychiatry 1987, 50:117-118; Lance J. W. Ann Neurol. 1977,2:285-293; and Nakano T., et al., Clin Neurol. 1982, 23:199-202; eachherein incorporated by reference in their entireties). The PDC attacksoccur both spontaneously while at rest and following provoking factorsthat include alcohol or caffeine consumption and to a lesser extentfatigue, hunger, and emotional stress.

A locus for autosomal dominant PDC on chromosome 2q33-2q35 has beenidentified. A consensus PDC locus interval spanning approximately 2.7 cMbetween DNA polymorphisms D2S295 and D2S163 has been identified (see,e.g., Fink J. K., et al., Am J Hum Genet. 1996, 59:140-145; Fouad G. T.,et al., Am J Hum Genet. 1996, 59:135-139; Jarman P. R., et al., Brain.1997, 120:2125-2130; Matsuo H., et al., Arch Neurol. 1999, 56:721-726;Hofele K., et al., Neurology. 1997, 49:1252-1257; Przuntek H., et al., JNeurol. 1983, 230:163-169; and Raskind W. H., et al., Hum Genet. 1998,102:93-97; Einum D. D., et al., Neurogenetics 1998, 1:289-292; GrunderS., et al., Eur J Hum Genet. 2001, 9:672-676; and Tokarz D., et al., AmJ Hum Genet. 2001, 69:629; each herein incorporated by reference intheir entireties). This region includes a cluster of ion channel genes(see, e.g., Fink J. K., et al., Am J Hum Genet. 1996, 59:140-145; hereinincorporated by reference in its entirety).

Presently, there is no cure for PDC. Medications used to treat PDCinclude anticonvulsant agents such as phenytoin, primidone, valporate,carbamazepine, phenobarbital, and diazepam, and anticholinergics,levodopa, flunarizine, and tetrabenazine. However, such medications areonly used to mask the symptoms of PDC.

What is needed is a better understanding of the pathophysiology,genetics and biochemistry underlying episodic movement disorders such asPDC. Additionally, better treatment options for PDC are needed, andimproved forms of diagnosis.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for research,diagnostic, drug screening, and therapeutic applications related to PDCand related conditions. In particular, the present invention providesmutations in the MR-1 gene associated with such conditions.

Accordingly, in some embodiments, the present invention provides anisolated and purified nucleic acid comprising a sequence encoding aprotein represented by SEQ ID NOs: 2, 3 or 4, and variants thereof. Inpreferred embodiments, the sequence is operably linked to a heterologouspromoter. In other preferred embodiments, the sequence is containedwithin a vector. In other preferred embodiments, the vector is within ahost cell.

In certain embodiments, the present invention provides a kit comprisinga reagent for detecting (e.g., reagents sufficient for detecting) thepresence or absence of a variant MR-1 polypeptide in a biologicalsample. In preferred embodiments, the kit further comprises instructionsfor using the kit for the detecting the presence or absence of a variantMR-1 polypeptide in a biological sample. In preferred embodiments, thereagent is one or more antibodies. In preferred embodiments, the variantMR-1 polypeptide contains an alanine to valine substitution at position7 and/or 9.

In certain embodiments, the present invention provides a method fordetection of a variant MR-1 polypeptide in a subject, comprising a)providing a biological sample from a subject, wherein the biologicalsample comprises a MR-1 polypeptide; and b) detecting the presence orabsence of a variant MR-1 polypeptide in the biological sample. Inpreferred embodiments, the variant MR-1 polypeptide contains an alanineto valine mismatch at position 7 and/or 9. In some embodiments, thebiological sample is selected from the group consisting of a bloodsample, a tissue sample, a urine sample, and an amniotic fluid sample,although the present invention is not limited to these embodiments.

In preferred embodiments, the subject is selected from the groupconsisting of an embryo, a fetus, a newborn animal, and a young animal.In other preferred embodiments, the detecting comprises differentialantibody binding. In other preferred embodiments, the detectioncomprises a Western blot. In yet other preferred embodiments, thedetecting comprises detecting a variant MR-1 nucleic acid sequence.

In certain embodiments, the present invention provides an isolated andpurified nucleic acid sequence that hybridizes under conditions of low,medium, or high stringency to a nucleic acid selected from the groupconsisting of SEQ ID NOs: 2, 3, and 4.

In certain embodiments, the present invention provides a vectorcomprising a nucleic acid sequence that hybridizes under conditions oflow, medium, or high stringency to a nucleic acid selected from thegroup consisting of SEQ ID NOs: 2, 3, and 4.

In certain embodiments, the present invention provides a host cellcomprising a vector comprising a nucleic acid sequence that hybridizesunder conditions of low, medium, or high stringency to a nucleic acidselected from the group consisting of SEQ ID NOs: 2, 3, and 4. Inpreferred embodiments, the host cell is located in an organism, whereinthe organism is a non-human animal.

In certain embodiments, the present invention provides a polypeptideencoded by a nucleic acid molecule containing a C66T mutation, whereinthe polypeptide is encoded by a nucleic acid selected from the groupconsisting of SEQ ID NOs: 2 and variants thereof that are at least 80%identical to SEQ ID NOs: 2. In preferred embodiments, the protein is atleast 90% or 95% identical to SEQ ID NO: 2.

In certain embodiments, the present invention provides a polypeptideencoded by a nucleic acid molecule containing a C72T mutation, whereinthe polypeptide is encoded by a nucleic acid selected from the groupconsisting of SEQ ID NOs: 3 and variants thereof that are at least 80%identical to SEQ ID NOs: 3. In preferred embodiments, the protein is atleast 90% or 95% identical to SEQ ID NO: 3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows wild type human MR-1 nucleic acid sequence (DNA) (SEQ IDNO: 1).

FIG. 2 shows a variant human MR-1 nucleic acid sequence (mRNA) (SEQ IDNO: 2).

FIG. 3 shows a variant human MR-1 nucleic acid sequence (mRNA) (SEQ IDNO: 3).

FIG. 4 shows a variant human MR-1 nucleic acid sequence (mRNA) (SEQ IDNO: 4).

FIG. 5 shows wild type human MR-1 amino acid sequence (SEQ ID NO: 5).

FIG. 6 shows a variant human MR-1 amino acid sequence (SEQ ID NO: 6).

FIG. 7 shows a variant human MR-1 amino acid sequence (SEQ ID NO: 7).

FIG. 8 shows a variant human MR-1 amino acid sequence (SEQ ID NO: 8).

FIG. 9 shows Paroxysmal dystonic choreoathetosis (PDC) kindreds linkedto the PDC locus on chromosome 2q33-2q35. Pedigrees of the PDC-Det(substitution of valine for alanine at amino acid position 9) and PDC-Pa(substitution of valine for alanine at amino acid position 7) kindredswith the myofibrillogenesis regulator 1 mutation (MR-1) are shown.Letters refer to MR-1 NM_(—)015488 complementary DNA sequence atnucleotides 72 (PDC-Det) and 66 (PDC-Pa). Open squares indicateunaffected men; open circles, unaffected women; closed squares, affectedmen; closed circles, affected women; question mark, affected-unaffectedstatus unknown; and slash, deceased.

FIG. 10 (SEQ ID NOS:11-14) shows myofibrillogenesis regulator 1mutations (MR-1) in subjects with paroxysmal dystonic choreoathetosis(PDC). Representative MR-1 sequence of normal and affected subjects fromPDC-Det (substitution of valine for alanine at amino acid position 9)and PDC-Pa (substitution of valine for alanine at amino acid position 7)kindreds are shown. The arrows mark the position of the MR-1NM_(—)015488 mutation in the PDC-Det (complementary DNA [cDNA]nucleotide C72T) and PDC-Pa (cDNA nucleotide C66T) kindreds.

FIG. 11 shows brain-specific expression of myofibrillogenesis regulator1 gene (MR-1) exon 1. A) Reverse transcription-polymerase chain reaction(RT-PCR) was used to amplify nucleotides 39 to 371 (333-base pair [bp]fragment) of the MR-1 NM_(—)015488 transcript from the human adultbrain, liver, kidney, skeletal muscle, heart, and lung messenger RNA(mRNA). B) The RT-PCR amplification of β-actin mRNA nucleotides 25 to650 (626-bp fragment) from the mRNA of these tissues.

FIG. 12 shows disease-specific myofibrillogenesis regulator 1 mutations(MR-1) A7V and A9V alter predicted a helix content of the MR-1amino-terminal region in paroxysmal dystonic choreoathetosis (PDC)kindreds PDC-Det (substitution of valine for alanine at amino acidposition 9) and PDC-Pa (substitution of valine for alanine at amino acidposition 7).

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “MR-1” when used in reference to a protein ornucleic acid refers to a MR-1 protein or nucleic acid encoding a MR-1protein of the present invention. The term MR-1 encompasses bothproteins that are identical to wild-type MR-1 and those that are derivedfrom wild type MR-1 (e.g., variants of MR-1 polypeptides of the presentinvention) or chimeric genes constructed with portions of MR-1 codingregions. In some embodiments, the “MR-1” is a wild type MR-1 nucleicacid (SEQ ID NO: 1) or amino acid sequence (SEQ ID NO: 5). In otherembodiments, the “MR-1” is a variant or mutant nucleic acid (SEQ ID NO:2, 3, and 4) or amino acid (SEQ ID NO: 6, 7, and 8).

As used herein, the terms “subject” and “patient” refer to any animal,such as a mammal like a dog, cat, bird, livestock, and preferably ahuman. Specific examples of “subjects” and “patients” include, but arenot limited to, individuals with PDC, and individuals with PDC-relatedcharacteristics or symptoms.

As used herein, the terms “episodic movement disorder,” “episodicneurologic disorder,” “paroxysmal disorder,” “paroxysmal neurologicaldisorder,” and the like, refer to neurological conditions sharing acommon symptom of involuntary movements. Examples of episodic movementdisorders include, but are not limited to, PDC, ataxia, bradykinesia,choreoathetosis, corticobasal degeneration, dyskinesias, dystonias,essential tremors, hereditary spastic paraplegia, Huntington's disease,multiple system atrophy, myoclonus, Parkinson's disease, progressivesupranuclear palsy, restless leg syndrome, Rett syndrome, spasticity,Sydenham's chorea, tardive dyskinesia, tics, Tourette's syndrome,tremor, and Wilson's disease.

As used herein, the phrase “symptoms of PDC” and “characteristics ofPDC” include, but are not limited to, attacks of involuntary movementscaused by a neurological dysfunction (e.g., dystonia, chorea, athetosis)lasting up to several hours and occuring at rest both spontaneously andfollowing caffeine or alcohol consumption.

The phrase “under conditions such that the symptoms are reduced” refersto any degree of qualitative or quantitative reduction in detectablesymptoms of PDC, including but not limited to, a detectable reduction onthe rate of recovery from PDC, or the reduction of at least one symptomof PDC.

The term “siRNAs” refers to short interfering RNAs. Methods for the useof siRNAs are described in U.S. Patent App. No.: 20030148519/A1 (hereinincorporated by reference). In some embodiments, siRNAs comprise aduplex, or double-stranded region, of about 18-25 nucleotides long;often siRNAs contain from about two to four unpaired nucleotides at the3′ end of each strand. At least one strand of the duplex ordouble-stranded region of a siRNA is substantially homologous to orsubstantially complementary to a target RNA molecule. The strandcomplementary to a target RNA molecule is the “antisense strand;” thestrand homologous to the target RNA molecule is the “sense strand,” andis also complementary to the siRNA antisense strand. siRNAs may alsocontain additional sequences; non-limiting examples of such sequencesinclude linking sequences, or loops, as well as stem and other foldedstructures. siRNAs appear to function as key intermediaries intriggering RNA interference in invertebrates and in vertebrates, and intriggering sequence-specific RNA degradation during posttranscriptionalgene silencing in plants.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing of gene expression by siRNAs. It is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector that is not integrated into the genome.The expression of the gene is either completely or partially inhibited.RNAi may also be considered to inhibit the function of a target RNA; thefunction of the target RNA may be complete or partial.

As used herein, the term “instructions for using said kit for saiddetecting the presence or absence of a variant MR-1 nucleic acid orpolypeptide in said biological sample” includes instructions for usingreagents contained in a kit for the detection of variant and wild typeMR-1 nucleic acids or polypeptides. In some embodiments, theinstructions further comprise the statement of intended use required bythe U.S. Food and Drug Administration (FDA) in labeling in vitrodiagnostic products.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, RNA (e.g., including but not limited to, mRNA, tRNA andrRNA) or precursor (e.g., MR-1). The polypeptide, RNA, or precursor canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction, etc.) ofthe full-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the sequences located adjacent tothe coding region on both the 5′ and 3′ ends for a distance of about 1kb on either end such that the gene corresponds to the length of thefull-length mRNA. The sequences that are located 5′ of the coding regionand which are present on the mRNA are referred to as 5′ untranslatedsequences. The sequences that are located 3′ or downstream of the codingregion and that are present on the mRNA are referred to as 3′untranslated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

In particular, the term “MR-1 gene” or “MR-1 genes” refers to thefull-length MR-1 nucleotide sequence (e.g., contained in SEQ ID NOs: 1,2, 3 and 4). However, it is also intended that the term encompassfragments of the MR-1 sequences, mutants of the MR-1 sequences, as wellas other domains within the full-length MR-1 nucleotide sequences.Furthermore, the terms “MR-1 nucleotide sequence” or “MR-1polynucleotide sequence” encompasses DNA sequences, cDNA sequences, RNA(e.g., mRNA) sequences, and associated regulatory sequences.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the terms“modified,” “mutant,” “polymorphism,” and “variant” refer to a gene orgene product that displays modifications in sequence and/or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides or polynucleotidesin a manner such that the 5′ phosphate of one mononucleotide pentosering is attached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage. Therefore, an end of an oligonucleotides orpolynucleotide, referred to as the “5′ end” if its 5′ phosphate is notlinked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequentmononucleotide pentose ring. As used herein, a nucleic acid sequence,even if internal to a larger oligonucleotide or polynucleotide, also maybe said to have 5′ and 3′ ends. In either a linear or circular DNAmolecule, discrete elements are referred to as being “upstream” or 5′ ofthe “downstream” or 3′ elements. This terminology reflects the fact thattranscription proceeds in a 5′ to 3′ fashion along the DNA strand. Thepromoter and enhancer elements that direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or, in other words, the nucleic acid sequencethat encodes a gene product. The coding region may be present in a cDNA,genomic DNA, or RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded (i.e., thesense strand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements include splicing signals,polyadenylation signals, termination signals, etc.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence5′-A-G-T-3′, is complementary to the sequence 3′-T-C-A-5′.Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids. Complementarity can include the formationof base pairs between any type of nucleotides, including non-naturalbases, modified bases, synthetic bases and the like.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The term “inhibition of binding,” when used in reference tonucleic acid binding, refers to inhibition of binding caused bycompetition of homologous sequences for binding to a target sequence.The inhibition of hybridization of the completely complementary sequenceto the target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous to a target under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target that lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “competes for binding” is used in reference toa first polypeptide with an activity which binds to the same substrateas does a second polypeptide with an activity, where the secondpolypeptide is a variant of the first polypeptide or a related ordissimilar polypeptide. The efficiency (e.g., kinetics orthermodynamics) of binding by the first polypeptide may be the same asor greater than or less than the efficiency substrate binding by thesecond polypeptide. For example, the equilibrium binding constant(K_(D)) for binding to the substrate may be different for the twopolypeptides. The term “K_(m)” as used herein refers to theMichaelis-Menton constant for an enzyme and is defined as theconcentration of the specific substrate at which a given enzyme yieldsone-half its maximum velocity in an enzyme catalyzed reaction.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with about 85-100% identity, preferably about70-100% identity). With medium stringency conditions, nucleic acid basepairing will occur between nucleic acids with an intermediate frequencyof complementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with about 50-70%identity). Thus, conditions of “weak” or “low” stringency are oftenrequired with nucleic acids that are derived from organisms that aregenetically diverse, as the frequency of complementary sequences isusually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5× Denhardt's reagent [50× Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

The present invention is not limited to the hybridization of probes ofabout 500 nucleotides in length. The present invention contemplates theuse of probes between approximately 10 nucleotides up to severalthousand (e.g., at least 5000) nucleotides in length. One skilled in therelevant understands that stringency conditions may be altered forprobes of other sizes (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985] and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY[1989]).

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “sequenceidentity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman [Smithand Waterman, Adv. Appl. Math. 2: 482 (1981)] by the homology alignmentalgorithm of Needleman and Wunsch [Needleman and Wunsch, J. Mol. Biol.48:443 (1970)], by the search for similarity method of Pearson andLipman [Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444(1988)], by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.),or by inspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term “sequence identity” means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a segment of the full-length sequences of thecompositions claimed in the present invention (e.g., MR-1).

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions that are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term “fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion as compared to thenative protein, but where the remaining amino acid sequence is identicalto the corresponding positions in the amino acid sequence deduced from afull-length cDNA sequence. Fragments typically are at least 4 aminoacids long, preferably at least 20 amino acids long, usually at least 50amino acids long or longer, and span the portion of the polypeptiderequired for intermolecular binding of the compositions (claimed in thepresent invention) with its various ligands and/or substrates.

The term “polymorphic locus” is a locus present in a population thatshows variation between members of the population (i.e., the most commonallele has a frequency of less than 0.95). In contrast, a “monomorphiclocus” is a genetic locus at little or no variations seen betweenmembers of the population (generally taken to be a locus at which themost common allele exceeds a frequency of 0.95 in the gene pool of thepopulation).

As used herein, the term “genetic variation information” or “geneticvariant information” refers to the presence or absence of one or morevariant nucleic acid sequences (e.g., polymorphism or mutations) in agiven allele of a particular gene (e.g., a MR-1 gene of the presentinvention).

As used herein, the term “detection assay” refers to an assay fordetecting the presence or absence of specific nucleic acid sequences(e.g., polymorphisms or mutations) in a given allele of a particulargene (e.g., a MR-1 gene).

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038[1972]). Other nucleic acid will not be replicated by this amplificationenzyme. Similarly, in the case of T7 RNA polymerase, this amplificationenzyme has a stringent specificity for its own promoters (Chamberlin etal., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzymewill not ligate the two oligonucleotides or polynucleotides, where thereis a mismatch between the oligonucleotide or polynucleotide substrateand the template at the ligation junction (D. Y. Wu and R. B. Wallace,Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue oftheir ability to function at high temperature, are found to display highspecificity for the sequences bounded and thus defined by the primers;the high temperature results in thermodynamic conditions that favorprimer hybridization with the target sequences and not hybridizationwith non-target sequences (H. A. Erlich (ed.), PCR Technology, StocktonPress [1989]).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template that may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.Particular examples of primers useful in the present invention include,but are not limited to, a primer of least 5 nucleotides from SEQ ID NOs:1, 2, 3 and 4, a primer of at least 10 nucleotides from SEQ ID NOs: 1,2, 3 and 4, a primer of at least 20 nucleotides from SEQ ID NOs: 1, 2, 3and 4, a primer of at least 30 nucleotides in length from SEQ ID NOs: 1,2, 3 and 4, a primer of at least 40 nucleotides in length from SEQ IDNOs: 1, 2, 3 and 4, a primer of at least 50 nucleotides in length fromSEQ ID NOs: 1, 2, 3 and 4, and a primer of at least 55 nucleotides inlength from SEQ ID NOs: 1, 2, 3 and 4.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” refers to a nucleic acid sequence orstructure to be detected or characterized. Thus, the “target” is soughtto be sorted out from other nucleic acid sequences. A “segment” isdefined as a region of nucleic acid within the target sequence.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule that is comprised of segments of DNA joinedtogether by means of molecular biological techniques.

As used herein, the term “antisense” is used in reference to RNAsequences that are complementary to a specific RNA sequence (e.g.,mRNA). Included within this definition are antisense RNA (“asRNA”)molecules involved in gene regulation by bacteria. Antisense RNA may beproduced by any method, including synthesis by splicing the gene(s) ofinterest in a reverse orientation to a viral promoter that permits thesynthesis of a coding strand. Once introduced into an embryo, thistranscribed strand combines with natural mRNA produced by the embryo toform duplexes. These duplexes then block either the furthertranscription of the mRNA or its translation. In this manner, mutantphenotypes may be generated. The term “antisense strand” is used inreference to a nucleic acid strand that is complementary to the “sense”strand. The designation (−) (i.e., “negative”) is sometimes used inreference to the antisense strand, with the designation (+) sometimesused in reference to the sense (i.e., “positive”) strand.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is present in a form or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA foundin the state they exist in nature. For example, a given DNA sequence(e.g., a gene) is found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, are found in the cell as a mixture withnumerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding MR-1 includes, by way of example, suchnucleic acid in cells ordinarily expressing MR-1 where the nucleic acidis in a chromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid, oligonucleotide, or polynucleotidemay be present in single-stranded or double-stranded form. When anisolated nucleic acid, oligonucleotide or polynucleotide is to beutilized to express a protein, the oligonucleotide or polynucleotidewill contain at a minimum the sense or coding strand (i.e., theoligonucleotide or polynucleotide may single-stranded), but may containboth the sense and anti-sense strands (i.e., the oligonucleotide orpolynucleotide may be double-stranded).

As used herein, a “portion of a chromosome” refers to a discrete sectionof the chromosome. Chromosomes are divided into sites or sections bycytogeneticists as follows: the short (relative to the centromere) armof a chromosome is termed the “p” arm; the long arm is termed the “q”arm. Each arm is then divided into 2 regions termed region 1 and region2 (region 1 is closest to the centromere). Each region is furtherdivided into bands. The bands may be further divided into sub-bands. Forexample, the 11p15.5 portion of human chromosome 11 is the portionlocated on chromosome 11 (11) on the short arm (p) in the first region(1) in the 5th band (5) in sub-band 5 (0.5). A portion of a chromosomemay be “altered;” for instance the entire portion may be absent due to adeletion or may be rearranged (e.g., inversions, translocations,expanded or contracted due to changes in repeat regions). In the case ofa deletion, an attempt to hybridize (i.e., specifically bind) a probehomologous to a particular portion of a chromosome could result in anegative result (i.e., the probe could not bind to the sample containinggenetic material suspected of containing the missing portion of thechromosome). Thus, hybridization of a probe homologous to a particularportion of a chromosome may be used to detect alterations in a portionof a chromosome.

The term “sequences associated with a chromosome” means preparations ofchromosomes (e.g., spreads of metaphase chromosomes), nucleic acidextracted from a sample containing chromosomal DNA (e.g., preparationsof genomic DNA); the RNA that is produced by transcription of geneslocated on a chromosome (e.g., hnRNA and mRNA), and cDNA copies of theRNA transcribed from the DNA located on a chromosome. Sequencesassociated with a chromosome may be detected by numerous techniquesincluding probing of Southern and Northern blots and in situhybridization to RNA, DNA, or metaphase chromosomes with probescontaining sequences homologous to the nucleic acids in the above listedpreparations.

As used herein the term “portion” when in reference to a nucleotidesequence (as in “a portion of a given nucleotide sequence”) refers tofragments of that sequence. The fragments may range in size from fournucleotides to the entire nucleotide sequence minus one nucleotide (10nucleotides, 20, 30, 40, 50, 100, 200, etc.).

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. The coding region is bounded, in eukaryotes, on the 5′side by the nucleotide triplet “ATG” that encodes the initiatormethionine and on the 3′ side by one of the three triplets, whichspecify stop codons (i.e., TAA, TAG, TGA).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, MR-1 antibodies are purifiedby removal of contaminating non-immunoglobulin proteins; they are alsopurified by the removal of immunoglobulin that does not bind a MR-1polypeptide. The removal of non-immunoglobulin proteins and/or theremoval of immunoglobulins that do not bind a MR-1 polypeptide resultsin an increase in the percent of MR-1-reactive immunoglobulins in thesample. In another example, recombinant MR-1 polypeptides are expressedin bacterial host cells and the polypeptides are purified by the removalof host cell proteins; the percent of recombinant MR-1 polypeptides isthereby increased in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

The term “native protein” as used herein, is used to indicate a proteinthat does not contain amino acid residues encoded by vector sequences;that is the native protein contains only those amino acids found in theprotein as it occurs in nature. A native protein may be produced byrecombinant means or may be isolated from a naturally occurring source.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp 9.31-9.58 [1989]).

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (J. Sambrook, et al., supra, pp 7.39-7.52[1989]).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies that bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

The term “transgene” as used herein refers to a foreign, heterologous,or autologous gene that is placed into an organism by introducing thegene into newly fertilized eggs or early embryos. The term “foreigngene” refers to any nucleic acid (e.g., gene sequence) that isintroduced into the genome of an animal by experimental manipulationsand may include gene sequences found in that animal so long as theintroduced gene does not reside in the same location as does thenaturally-occurring gene. The term “autologous gene” is intended toencompass variants (e.g., polymorphisms or mutants) of the naturallyoccurring gene. The term transgene thus encompasses the replacement ofthe naturally occurring gene with a variant form of the gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher than that typically observedin a given tissue in a control or non-transgenic animal. Levels of mRNAare measured using any of a number of techniques known to those skilledin the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the RAD50mRNA-specific signal observed on Northern blots). The amount of mRNApresent in the band corresponding in size to the correctly spliced MR-1transgene RNA is quantified; other minor species of RNA which hybridizeto the transgene probe are not considered in the quantification of theexpression of the transgenic mRNA.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

A “composition comprising a given polynucleotide sequence” as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution. Compositions comprising polynucleotide sequences encodingMR-1s (e.g., SEQ ID NOs: 1, 2, 3 and 4) or fragments thereof may beemployed as hybridization probes. In this case, the MR-1 encodingpolynucleotide sequences are typically employed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., SDS), and othercomponents (e.g., Denhardt's solution, dry milk, salmon sperm DNA,etc.).

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the screeningmethods of the present invention. A “known therapeutic compound” refersto a therapeutic compound that has been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in such treatment or prevention.

The term “sample” as used herein is used in its broadest sense. A samplesuspected of containing a human chromosome or sequences associated witha human chromosome may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern blot analysis), RNA (insolution or bound to a solid support such as for Northern blotanalysis), cDNA (in solution or bound to a solid support) and the like.A sample suspected of containing a protein may comprise a cell, aportion of a tissue, an extract containing one or more proteins and thelike.

As used herein, the term “response,” when used in reference to an assay,refers to the generation of a detectable signal (e.g., accumulation ofreporter protein, increase in ion concentration, accumulation of adetectable chemical product).

As used herein, the term “reporter gene” refers to a gene encoding aprotein that may be assayed. Examples of reporter genes include, but arenot limited to, luciferase (See, e.g., deWet et al., Mol. Cell. Biol.7:725 [1987] and U.S. Pat. Nos., 6,074,859; 5,976,796; 5,674,713; and5,618,682; all of which are incorporated herein by reference), greenfluorescent protein (e.g., GenBank Accession Number U43284; a number ofGFP variants are commercially available from CLONTECH Laboratories, PaloAlto, Calif.), chloramphenicol acetyltransferase, β-galactosidase,alkaline phosphatase, and horse radish peroxidase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the MR-1 proteins and nucleic acidsencoding MR-1 proteins. The present invention further provides assaysfor the identification of therapeutic agents, and for the detection ofMR-1 polymorphisms and mutations associated with disease states.Exemplary embodiments of the present invention are described below.

Individuals with PDC are normal until they have “attacks” of involuntarymovements that may last for 15 minutes to several hours. These “attacks”of involuntary movements occur spontaneously at rest without obviousprovocation and also following consumption of alcohol and caffeine.

Two missense MR-1 mutations (e.g., SEQ ID NOs: 2 (C66T) and 3 (C72T)were identified within the 2q33-2q35 chromosomal locus as segregatingwith individuals suffering from PDC and not segregating with controlindividuals (as described in Examples 1-11). These mutations predictsubstitutions of valine for alanine at residue 9 (A9V) and residue 7(A7V). The alanine residues at positions 7 and 9 are part of anamino-terminal α helix that becomes disrupted with either valinesubstitution. The 7 Ala and 9 Ala residues are conserved in the 2species (human and mouse) for which the MR-1 cDNA sequence is known andin the amino acid sequence deduced from rat genomic DNA (see, e.g.,contig NW_(—)047816).

MR-1 sequences are present in 2 transcripts, NM_(—)015488 andNM_(—)022573, that share 8 exons but differ in the first 2 exons. TheMR-1 transcript NM_(—)015488 (derived from 10 exons spanning 76.3 kb ofgenomic DNA) encodes a 385 amino acid protein of 42.9 kDa (predicted).The MR-1 transcript NM_(—)022573 (derived from 9 exons spanning 23.5 kbof genomic DNA) encodes a 361 amino acid protein of 40.7 kDa(predicted). Only the MR-1 transcript NM_(—)015488 contained exon 1. TheMR-1 NM_(—)015488 transcript containing exon 1 is expressed only in thebrain. The PDC-specific MR-1 NM_(—)015488 mutations C66T and C72T arepresent in exon 1. That this exon is expressed only in the brain couldexplain why PDC symptoms are restricted to this area.

Discovery of the cause of PDC a) provides laboratory-based and clinicalbased diagnostic testing for the disorder; b) provides methods ofidentifying and characterizing treatments for the disorder; c) provideinsights into the causes and treatments for other episodic movementdisorders including dystonias, Parkinson's disease, Tics, Tourette'ssyndrome, choreas including Huntington's chorea, drug-induced movementdisorders including neuroleptic induced tardive dyskinesia; and d)provide insights into the neurophysiologic effects of alcohol andcaffeine on the nervous system.

MR-1 gene sequence analysis can be used to diagnose PDC in an individualand distinguish this condition from other episodic and non-episodicmovement disorders. Knowledge that a MR-1 pathway disturbance and/orMR-1 gene mutation results in PDC can be applied directly to genetictesting to diagnose this disease; provide genetic counseling for thisdisease; or to diagnose related episodic movement disorders

Genetic and/or enzyme testing can indicate individual sensitivity todrug-induced movement disorders. Attacks of involuntary movements in PDCare often triggered by specific drugs (alcohol and caffeine).Drug-induced movement disorders are a common problem in medicine,particularly with the use of neuroleptic medications (including, e.g.,haloperidol and thioridazine) in which the appearance of drug-inducedabnormal movements (“tardive dyskinesia”) may cause profound andpermanent impairment. Discovery that MR-1 gene mutations cause adrug-induced movement disorder provides insight into this process forother types of drug-induced movement disorders. This informationprovides insight into the causes and treatments for these other types ofdrug-induced movement disorders.

Knowledge that genetic variation in MR-1 and the MR-1 pathway causehuman neurologic disease, combined with knowledge that symptoms relatedto PDC are exacerbated following alcohol or caffeine consumption can beused as the basis for genetic testing to determine human and animalvulnerability to the neurotoxic effects of alcohol and caffeine or otherdrugs that cause involuntary movements. One application is the screeningof individuals taking neuroleptic medications for risk of developingdrug induced involuntary movements.

Knowledge that genetic variation in MR-1 and the MR-1 pathway causehuman neurologic disease (e.g., PDC) provides therapeutic pathways(including but not limited to MR-1 protein or gene replacement) forthese disorders.

Knowledge that genetic variation in the MR-1 pathway and MR-1 genemutations cause involuntary movements can be used as the basis fortreatment of episodic movement disorders (including, but not limited to,dystonias, choreas, tremor, tics, Tourette's drug-induced movementdisorders, and other paroxysmal neurologic diseases including epilepsy,restless leg syndrome, migraine, episodic dystonias, episodic ataxias).Such treatments include (but are not limited to) replacement of MR-1biochemical function either as small molecule biochemical intermediateor protein (such as enzyme) or gene replacement strategies.

The present invention is described in more detail in the followingsections: I. MR-1 Polynucleotides, II. MR-1 Polypeptides, III. Detectionof MR-1 Alleles IV. Generation of MR-1 Antibodies, V. Gene Therapy UsingMR-1, VI. Transgenic Animals Expressing Exogenous MR-1 Genes andHomologs, Mutants, and Variants Thereof, VII. Drug Screening Using MR-1,VIII. Pharmaceutical Compositions Containing MR-1 Nucleic Acid,Peptides, and Analogs, and IX. RNAi for MR-1.

I. MR-1 Polynucleotides

As described above, the present invention provides novel MR-1 familygenes. Accordingly, the present invention provides nucleic acidsencoding MR-1 genes, homologs, variants (e.g., polymorphisms andmutants), including but not limited to, those described in SEQ ID NOs:1, 2, 3 and 4. Table 1 describes exemplary MR-1 genes of the presentinvention. In some embodiments, the present invention providespolynucleotide sequences that are capable of hybridizing to SEQ ID NOs:1, 2, 3 and 4 under conditions of low to high stringency as long as thepolynucleotide sequence capable of hybridizing encodes a protein thatretains a biological activity of the naturally occurring MR-1s. In someembodiments, the protein that retains a biological activity of naturallyoccurring MR-1 is 70% homologous to wild-type MR-1, preferably 80%homologous to wild-type MR-1, more preferably 90% homologous towild-type MR-1, and most preferably 95% homologous to wild-type MR-1. Inpreferred embodiments, hybridization conditions are based on the meltingtemperature (T_(m)) of the nucleic acid binding complex and confer adefined “stringency” as explained above (See e.g., Wahl, et al., Meth.Enzymol., 152:399-407 [1987], incorporated herein by reference).

In other embodiments of the present invention, additional alleles ofMR-1 genes are provided. In preferred embodiments, alleles result from apolymorphism or mutation (i.e., a change in the nucleic acid sequence)and generally produce altered mRNAs or polypeptides whose structure orfunction may or may not be altered. Any given gene may have none, one ormany allelic forms. Common mutational changes that give rise to allelesare generally ascribed to deletions, additions or substitutions ofnucleic acids. Each of these types of changes may occur alone, or incombination with the others, and at the rate of one or more times in agiven sequence. Examples of the alleles of the present invention includethat encoded by SEQ ID NO: 1 (wild type) and disease alleles thereof(e.g., SEQ ID NOs: 2, 3 and 4). Additional examples include truncationmutations (e.g., such that the encoded mRNA does not produce a completeprotein). Mutations or sequences that are in linkage disequilibrium withmutations described herein may also be detected as a surrogate fordetecting the mutations directly.

In still other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alteran MR-1 coding sequence for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing and/orexpression of the gene product. For example, mutations may be introducedusing techniques that are well known in the art (e.g., site-directedmutagenesis to insert new restriction sites, to alter glycosylationpatterns, to change codon preference, etc.).

In some embodiments of the present invention, the polynucleotidesequence of MR-1 may be extended utilizing the nucleotide sequence invarious methods known in the art to detect upstream sequences such aspromoters and regulatory elements. For example, it is contemplated thatrestriction-site polymerase chain reaction (PCR) will find use in thepresent invention. This is a direct method that uses universal primersto retrieve unknown sequence adjacent to a known locus (Gobinda et al.,PCR Methods Applic., 2:318-22 [1993]). First, genomic DNA is amplifiedin the presence of a primer to a linker sequence and a primer specificto the known region. The amplified sequences are then subjected to asecond round of PCR with the same linker primer and another specificprimer internal to the first one. Products of each round of PCR aretranscribed with an appropriate RNA polymerase and sequenced usingreverse transcriptase.

In another embodiment, inverse PCR can be used to amplify or extendsequences using divergent primers based on a known region (Triglia etal., Nucleic Acids Res., 16:8186 [1988]). The primers may be designedusing Oligo 4.0 (National Biosciences Inc, Plymouth Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68-72° C. The method uses several restriction enzymesto generate a suitable fragment in the known region of a gene. Thefragment is then circularized by intramolecular ligation and used as aPCR template. In still other embodiments, walking PCR is utilized.Walking PCR is a method for targeted gene walking that permits retrievalof unknown sequence (Parker et al., Nucleic Acids Res., 19:3055-60[1991]).

The PROMOTERFINDER kit (Clontech) uses PCR, nested primers and speciallibraries to “walk in” genomic DNA. This process avoids the need toscreen libraries and is useful in finding intron/exon junctions.

Preferred libraries for screening for full length cDNAs includemammalian libraries that have been size-selected to include largercDNAs. Also, random primed libraries are preferred, in that they willcontain more sequences that contain the 5′ and upstream gene regions. Arandomly primed library may be particularly useful in case where anoligo d(T) library does not yield full-length cDNA. Genomic mammalianlibraries are useful for obtaining introns and extending 5′ sequence.

In other embodiments of the present invention, variants of the disclosedMR-1 sequences are provided (e.g., SEQ ID NOs: 2, 3 and 4). In preferredembodiments, variants result from polymorphisms or mutations (i.e., achange in the nucleic acid sequence) and generally produce altered mRNAsor polyp eptides whose structure or function may or may not be altered.Any given gene may have none, one, or many variant forms. Commonmutational changes that give rise to variants are generally ascribed todeletions, additions or substitutions of nucleic acids. Each of thesetypes of changes may occur alone, or in combination with the others, andat the rate of one or more times in a given sequence.

It is contemplated that it is possible to modify the structure of apeptide having a function (e.g., MR-1 function) for such purposes asaltering the biological activity (e.g., altered MR-1 function). Suchmodified pepti des are considered functional equivalents of peptideshaving an activity of a MR-1 peptide as defined herein. A modifiedpeptide can be produced in which the nucleotide sequence encoding thepolypeptide has been altered, such as by substitution, deletion, oraddition. In particularly preferred embodiments, these modifications donot significantly reduce the biological activity of the modified MR-1genes. In other words, construct “X” can be evaluated in order todetermine whether it is a member of the genus of modified or variantMR-1's of the present invention as defined functionally, rather thanstructurally. In preferred embodiments, the activity of variant MR-1polypeptides is evaluated by methods described herein (e.g., thegeneration of transgenic animals or the use of signaling assays).

Moreover, as described above, variant forms of MR-1 genes are alsocontemplated as being equivalent to those peptides and DNA moleculesthat are set forth in more detail herein. For example, it iscontemplated that isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid (i.e., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Accordingly, someembodiments of the present invention provide variants of MR-1 disclosedherein containing conservative replacements. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Genetically encoded amino acids can bedivided into four families: (1) acidic (aspartate, glutamate); (2) basic(lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17-21, 2nd ed, WH Freeman and Co., 1981). Whether achange in the amino acid sequence of a peptide results in a functionalpolypeptide can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner.

More rarely, a variant includes “nonconservative” changes (e.g.,replacement of a glycine with a tryptophan). Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs (e.g., LASERGENE software, DNASTAR Inc., Madison,Wis.).

As described in more detail below, variants may be produced by methodssuch as directed evolution or other techniques for producingcombinatorial libraries of variants, described in more detail below. Instill other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alter aMR-1 coding sequence including, but not limited to, alterations thatmodify the cloning, processing, localization, secretion, and/orexpression of the gene product. For example, mutations may be introducedusing techniques that are well known in the art (e.g., site-directedmutagenesis to insert new restriction sites, alter glycosylationpatterns, or change codon preference, etc.).

TABLE 1 MR-1 Genes SEQ ID NO SEQ ID NO MR-1 Gene (Nucleic acid)(Polypeptide) MR-1 1 5 MR-1 (C51T) 2 6 MR-1 (C57T) 3 7 MR-1 (C51T)(C57T) 4 8II. MR-1 Polypeptides

In other embodiments, the present invention provides MR-1 polynucleotidesequences that encode MR-1 polypeptide sequences (e.g., the polypeptidesof SEQ ID NOs: 5, 6, 7 and 8). Other embodiments of the presentinvention provide fragments, fusion proteins or functional equivalentsof these MR-1 proteins. In some embodiments, the present inventionprovides mutants of MR-1 polypeptides. In still other embodiments of thepresent invention, nucleic acid sequences corresponding to MR-1variants, homologs, and mutants may be used to generate recombinant DNAmolecules that direct the expression of the MR-1 variants, homologs, andmutants in appropriate host cells. In some embodiments of the presentinvention, the polypeptide may be a naturally purified product, in otherembodiments it may be a product of chemical synthetic procedures, and instill other embodiments it may be produced by recombinant techniquesusing a prokaryotic or eukaryotic host (e.g., by bacterial, yeast,higher plant, insect and mammalian cells in culture). In someembodiments, depending upon the host employed in a recombinantproduction procedure, the polypeptide of the present invention may beglycosylated or may be non-glycosylated. In other embodiments, thepolypeptides of the invention may also include an initial methionineamino acid residue.

In one embodiment of the present invention, due to the inherentdegeneracy of the genetic code, DNA sequences other than thepolynucleotide sequences of SEQ ID NOs: 1, 2, 3 and 4 that encodesubstantially the same or a functionally equivalent amino acid sequence,may be used to clone and express MR-1. In general, such polynucleotidesequences hybridize to SEQ ID NOs: 1, 2, 3 and 4 under conditions ofhigh to medium stringency as described above. As will be understood bythose of skill in the art, it may be advantageous to produce MR-1encoding nucleotide sequences possessing non-naturally occurring codons.Therefore, in some preferred embodiments, codons preferred by aparticular prokaryotic or eukaryotic host (Murray et al., Nucl. AcidsRes., 17 [1989]) are selected, for example, to increase the rate of MR-1expression or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, than transcripts produced fromnaturally occurring sequence.

1. Vectors for Production of MR-1

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. In some embodiments of the presentinvention, vectors include, but are not limited to, chromosomal,nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40,bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, and viral DNA suchas vaccinia, adenovirus, fowl pox virus, and pseudorabies). It iscontemplated that any vector may be used as long as it is replicable andviable in the host.

In particular, some embodiments of the present invention providerecombinant constructs comprising one or more of the sequences asbroadly described above (e.g., SEQ ID NOs: 1, 2, 3 and 4). In someembodiments of the present invention, the constructs comprise a vector,such as a plasmid or viral vector, into which a sequence of theinvention has been inserted, in a forward or reverse orientation. Instill other embodiments, the heterologous structural sequence (e.g., SEQID NOs: 1, 2, 3 and 4) is assembled in appropriate phase withtranslation initiation and termination sequences. In preferredembodiments of the present invention, the appropriate DNA sequence isinserted into the vector using any of a variety of procedures. Ingeneral, the DNA sequence is inserted into an appropriate restrictionendonuclease site(s) by procedures known in the art.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. Such vectors include, but are notlimited to, the following vectors: 1) Bacterial—pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, PBSKS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44, PXT1,pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); and 3)Baculovirus—pPbac and pMbac (Stratagene). Any other plasmid or vectormay be used as long as they are replicable and viable in the host. Insome preferred embodiments of the present invention, mammalianexpression vectors comprise an origin of replication, a suitablepromoter and enhancer, and also any necessary ribosome binding sites,polyadenylation sites, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences. Inother embodiments, DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

In certain embodiments of the present invention, the DNA sequence in theexpression vector is operatively linked to an appropriate expressioncontrol sequence(s) (promoter) to direct mRNA synthesis. Promotersuseful in the present invention include, but are not limited to, the LTRor SV40 promoter, the E. coli lac or trp, the phage lambda P_(L) andP_(R), T3 and T7 promoters, and the cytomegalovirus (CMV) immediateearly, herpes simplex virus (HSV) thymidine kinase, and mousemetallothionein-I promoters and other promoters known to controlexpression of genes in prokaryotic or eukaryotic cells or their viruses.In other embodiments of the present invention, recombinant expressionvectors include origins of replication and selectable markers permittingtransformation of the host cell (e.g., dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, or tetracycline orampicillin resistance in E. coli).

In some embodiments of the present invention, transcription of the DNAencoding the polypeptides of the present invention by higher eukaryotesis increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp that act on a promoter to increase its transcription. Enhancersuseful in the present invention include, but are not limited to, theSV40 enhancer on the late side of the replication origin bp 100 to 270,a cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

In other embodiments, the expression vector also contains a ribosomebinding site for translation initiation and a transcription terminator.In still other embodiments of the present invention, the vector may alsoinclude appropriate sequences for amplifying expression.

2. Host Cells for Production of MR-1 Polypeptides

In a further embodiment, the present invention provides host cellscontaining the above-described constructs. In some embodiments of thepresent invention, the host cell is a higher eukaryotic cell (e.g., amammalian or insect cell). In other embodiments of the presentinvention, the host cell is a lower eukaryotic cell (e.g., a yeastcell). In still other embodiments of the present invention, the hostcell can be a prokaryotic cell (e.g., a bacterial cell). Specificexamples of host cells include, but are not limited to, Escherichiacoli, Salmonella typhimurium, Bacillus subtilis, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus, as wellas Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila S2cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells, COS-7lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175 [1981]), C127,3T3, 293, 293T, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. In someembodiments, introduction of the construct into the host cell can beaccomplished by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (See e.g., Davis et al., Basic Methodsin Molecular Biology, [1986]). Alternatively, in some embodiments of thepresent invention, the polypeptides of the invention can besynthetically produced by conventional peptide synthesizers.

Proteins can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the present invention. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., [1989].

In some embodiments of the present invention, following transformationof a suitable host strain and growth of the host strain to anappropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. In other embodiments of thepresent invention, cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. In still other embodiments of thepresent invention, microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

3. Purification of MR-1 polypeptides

The present invention also provides methods for recovering and purifyingMR-1 polypeptides from recombinant cell cultures including, but notlimited to, ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. In other embodiments of the present invention,protein-refolding steps can be used as necessary, in completingconfiguration of the mature protein. In still other embodiments of thepresent invention, high performance liquid chromatography (HPLC) can beemployed for final purification steps.

The present invention further provides polynucleotides having a codingsequence of a MR-1 gene (e.g., SEQ ID NOs: 1, 2, 3 and 4) fused in frameto a marker sequence that allows for purification of the polypeptide ofthe present invention. A non-limiting example of a marker sequence is ahexahistidine tag which may be supplied by a vector, preferably a pQE-9vector, which provides for purification of the polypeptide fused to themarker in the case of a bacterial host, or, for example, the markersequence may be a hemagglutinin (HA) tag when a mammalian host (e.g.,COS-7 cells) is used. The HA tag corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell, 37:767[1984]).

4. Truncation Mutants of MR-1 Polypeptide

In addition, the present invention provides fragments of MR-1polypeptides (i.e., truncation mutants). In some embodiments of thepresent invention, when expression of a portion of the MR-1 protein isdesired, it may be necessary to add a start codon (ATG) to theoligonucleotide fragment containing the desired sequence to beexpressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al., J. Bacteriol., 169:751 [1987]) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al., Proc. Natl. Acad. Sci. USA 84:2718[1990]). Therefore, removal of an N-terminal methionine, if desired, canbe achieved either in vivo by expressing such recombinant polypeptidesin a host which produces MAP (e.g., E. coli or CM89 or S. cerivisiae),or in vitro by use of purified MAP.

5. Fusion Proteins Containing MR-1

The present invention also provides fusion proteins incorporating all orpart of the MR-1 polypeptides of the present invention. Accordingly, insome embodiments of the present invention, the coding sequences for thepolypeptide can be incorporated as a part of a fusion gene including anucleotide sequence encoding a different polypeptide. It is contemplatedthat this type of expression system will find use under conditions whereit is desirable to produce an immunogenic fragment of a MR-1 protein. Insome embodiments of the present invention, the VP6 capsid protein ofrotavirus is used as an immunologic carrier protein for portions of aMR-1 polypeptide, either in the monomeric form or in the form of a viralparticle. In other embodiments of the present invention, the nucleicacid sequences corresponding to the portion of a MR-1 polypeptideagainst which antibodies are to be raised can be incorporated into afusion gene construct which includes coding sequences for a latevaccinia virus structural protein to produce a set of recombinantviruses expressing fusion proteins comprising a portion of MR-1 as partof the virion. It has been demonstrated with the use of immunogenicfusion proteins utilizing the hepatitis B surface antigen fusionproteins that recombinant hepatitis B virions can be utilized in thisrole as well. Similarly, in other embodiments of the present invention,chimeric constructs coding for fusion proteins containing a portion of aMR-1 polypeptide and the poliovirus capsid protein are created toenhance immunogenicity of the set of polypeptide antigens (See e.g., EPPublication No. 025949; and Evans et al., Nature 339:385 [1989]; Huanget al., J. Virol., 62:3855 [1988]; and Schlienger et al., J. Virol.,66:2 [1992]).

In still other embodiments of the present invention, the multipleantigen peptide system for peptide-based immunization can be utilized.In this system, a desired portion of MR-1 is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see e.g., Posnett et al., J. Biol. Chem., 263:1719 [1988];and Nardelli et al., J. Immunol., 148:914 [1992]). In other embodimentsof the present invention, antigenic determinants of the MR-1 proteinscan also be expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, such as a MR-1 protein of the present invention.Accordingly, in some embodiments of the present invention, MR-1polypeptides can be generated as glutathione-S-transferase (i.e., GSTfusion proteins). It is contemplated that such GST fusion proteins willenable easy purification of MR-1 polypeptides, such as by the use ofglutathione-derivatized matrices (See e.g., Ausabel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, NY [1991]).In another embodiment of the present invention, a fusion gene coding fora purification leader sequence, such as a poly-(His)/enterokinasecleavage site sequence at the N-terminus of the desired portion of aMR-1 polypeptide, can allow purification of the expressed MR-1 fusionprotein by affinity chromatography using a Ni²⁺ metal resin. In stillanother embodiment of the present invention, the purification leadersequence can then be subsequently removed by treatment with enterokinase(See e.g., Hochuli et al., J. Chromatogr., 411:177 [1987]; and Janknechtet al., Proc. Natl. Acad. Sci. USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment ofthe present invention, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, in other embodiments of the present invention, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed to generate a chimeric genesequence (See e.g., Current Protocols in Molecular Biology, supra).

6. Variants of MR-1

Still other embodiments of the present invention provide mutant orvariant forms of MR-1 polypeptides (i.e., muteins). It is possible tomodify the structure of a peptide having an activity of a MR-1polypeptide of the present invention for such purposes as enhancingtherapeutic or prophylactic efficacy, disabling the protein, orstability (e.g., ex vivo shelf life, and/or resistance to proteolyticdegradation in vivo). Such modified peptides are considered functionalequivalents of peptides having an activity of the subject MR-1 proteinsas defined herein. A modified peptide can be produced in which the aminoacid sequence has been altered, such as by amino acid substitution,deletion, or addition.

Moreover, as described above, variant forms (e.g., mutants orpolymorphic sequences) of the subject MR-1 proteins are alsocontemplated as being equivalent to those peptides and DNA moleculesthat are set forth in more detail. For example, as described above, thepresent invention encompasses mutant and variant proteins that containconservative or non-conservative amino acid substitutions.

This invention further contemplates a method of generating sets ofcombinatorial mutants of the present MR-1 proteins, as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (i.e., mutants or polymorphic sequences) that areinvolved in episodic movement disorders (e.g., PDC) or resistance toepisodic movement disorders. The purpose of screening such combinatoriallibraries is to generate, for example, novel MR-1 variants that can actas either agonists or antagonists, or alternatively, possess novelactivities all together.

Therefore, in some embodiments of the present invention, MR-1 variantsare engineered by the present method to provide altered (e.g., increasedor decreased) biological activity. In other embodiments of the presentinvention, combinatorially-derived variants are generated which have aselective potency relative to a naturally occurring MR-1. Such proteins,when expressed from recombinant DNA constructs, can be used in genetherapy protocols.

Still other embodiments of the present invention provide MR-1 variantsthat have intracellular half-lives dramatically different than thecorresponding wild-type protein. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular process that result in destruction of, or otherwiseinactivate MR-1 polypeptides. Such variants, and the genes which encodethem, can be utilized to alter the location of MR-1 expression bymodulating the half-life of the protein. For instance, a short half-lifecan give rise to more transient MR-1 biological effects and, when partof an inducible expression system, can allow tighter control of MR-1levels within the cell. As above, such proteins, and particularly theirrecombinant nucleic acid constructs, can be used in gene therapyprotocols.

In still other embodiments of the present invention, MR-1 variants aregenerated by the combinatorial approach to act as antagonists, in thatthey are able to interfere with the ability of the correspondingwild-type protein to regulate cell function.

In some embodiments of the combinatorial mutagenesis approach of thepresent invention, the amino acid sequences for a population of MR-1homologs, variants or other related proteins are aligned, preferably topromote the highest homology possible. Such a population of variants caninclude, for example, MR-1 homologs from one or more species, or MR-1variants from the same species but which differ due to mutation orpolymorphisms. Amino acids that appear at each position of the alignedsequences are selected to create a degenerate set of combinatorialsequences.

In a preferred embodiment of the present invention, the combinatorialMR-1 library is produced by way of a degenerate library of genesencoding a library of polypeptides which each include at least a portionof potential MR-1 protein sequences. For example, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential MR-1 sequences are expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of MR-1 sequencestherein.

There are many ways by which the library of potential MR-1 homologs andvariants can be generated from a degenerate oligonucleotide sequence. Insome embodiments, chemical synthesis of a degenerate gene sequence iscarried out in an automatic DNA synthesizer, and the synthetic genes areligated into an appropriate gene for expression. The purpose of adegenerate set of genes is to provide, in one mixture, all of thesequences encoding the desired set of potential MR-1 sequences. Thesynthesis of degenerate oligonucleotides is well known in the art (Seee.g., Narang, Tetrahedron Lett., 39:39 [1983]; Itakura et al.,Recombinant DNA, in Walton (ed.), Proceedings of the 3rd ClevelandSymposium on Macromolecules, Elsevier, Amsterdam, pp 273-289 [1981];Itakura et al., Annu. Rev. Biochem., 53:323 [1984]; Itakura et al.,Science 198:1056 [1984]; Ike et al., Nucl. Acid Res., 11:477 [1983]).Such techniques have been employed in the directed evolution of otherproteins (See e.g., Scott et al., Science 249:386 [1980]; Roberts etal., Proc. Natl. Acad. Sci. USA 89:2429 [1992]; Devlin et al., Science249: 404 [1990]; Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378[1990]; each of which is herein incorporated by reference; as well asU.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815; each of which isincorporated herein by reference).

It is contemplated that the MR-1 nucleic acids of the present invention(e.g., SEQ ID NOs: 1, 2, 3 and 4, and fragments and variants thereof)can be utilized as starting nucleic acids for directed evolution. Thesetechniques can be utilized to develop MR-1 variants having desirableproperties such as increased or decreased biological activity.

In some embodiments, artificial evolution is performed by randommutagenesis (e.g., by utilizing error-prone PCR to introduce randommutations into a given coding sequence). This method requires that thefrequency of mutation be finely tuned. As a general rule, beneficialmutations are rare, while deleterious mutations are common. This isbecause the combination of a deleterious mutation and a beneficialmutation often results in an inactive enzyme. The ideal number of basesubstitutions for targeted gene is usually between 1.5 and 5 (Moore andArnold, Nat. Biotech., 14, 458 [1996]; Leung et al., Technique, 1:11[1989]; Eckert and Kunkel, PCR Methods Appl., 1:17-24 [1991]; Caldwelland Joyce, PCR Methods Appl., 2:28 [1992]; and Zhao and Arnold, Nuc.Acids. Res., 25:1307 [1997]). After mutagenesis, the resulting clonesare selected for desirable activity (e.g., screened for MR-1 activity).Successive rounds of mutagenesis and selection are often necessary todevelop enzymes with desirable properties. It should be noted that onlythe useful mutations are carried over to the next round of mutagenesis.

In other embodiments of the present invention, the polynucleotides ofthe present invention are used in gene shuffling or sexual PCRprocedures (e.g., Smith, Nature, 370:324 [1994]; U.S. Pat. Nos.5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are hereinincorporated by reference). Gene shuffling involves random fragmentationof several mutant DNAs followed by their reassembly by PCR into fulllength molecules. Examples of various gene shuffling procedures include,but are not limited to, assembly following DNase treatment, thestaggered extension process (STEP), and random priming in vitrorecombination. In the DNase mediated method, DNA segments isolated froma pool of positive mutants are cleaved into random fragments with DNaseIand subjected to multiple rounds of PCR with no added primer. Thelengths of random fragments approach that of the uncleaved segment asthe PCR cycles proceed, resulting in mutations in present in differentclones becoming mixed and accumulating in some of the resultingsequences. Multiple cycles of selection and shuffling have led to thefunctional enhancement of several enzymes (Stemmer, Nature, 370:398[1994]; Stemmer, Proc. Natl. Acad. Sci. USA, 91:10747 [1994]; Crameri etal., Nat. Biotech., 14:315 [1996]; Zhang et al., Proc. Natl. Acad. Sci.USA, 94:4504 [1997]; and Crameri et al., Nat. Biotech., 15:436 [1997]).Variants produced by directed evolution can be screened for MR-1activity by the methods described herein.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques will be generally adaptable for rapid screening of thegene libraries generated by the combinatorial mutagenesis orrecombination of MR-1 homologs or variants. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected.

7. Chemical Synthesis of MR-1 Polypeptides

In an alternate embodiment of the invention, the coding sequence of MR-1is synthesized, whole or in part, using chemical methods well known inthe art (See e.g., Caruthers et al., Nucl. Acids Res. Symp. Ser., 7:215[1980]; Crea and Horn, Nucl. Acids Res., 9:2331 [1980]; Matteucci andCaruthers, Tetrahedron Lett., 21:719 [1980]; and Chow and Kempe, Nucl.Acids Res., 9:2807 [1981]). In other embodiments of the presentinvention, the protein itself is produced using chemical methods tosynthesize either an entire MR-1 amino acid sequence or a portionthereof. For example, peptides can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (See e.g., Creighton, ProteinsStructures And Molecular Principles, W H Freeman and Co, New York N.Y.[1983]). In other embodiments of the present invention, the compositionof the synthetic peptides is confirmed by amino acid analysis orsequencing (See e.g., Creighton, supra).

Direct peptide synthesis can be performed using various solid-phasetechniques (Roberge et al., Science 269:202 [1995]) and automatedsynthesis may be achieved, for example, using ABI 431 A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer. Additionally, the amino acid sequence of a MR-1polypeptide, or any part thereof, may be altered during direct synthesisand/or combined using chemical methods with other sequences to produce avariant polypeptide.

III. Detection of MR-1 Alleles

In some embodiments, the present invention provides methods of detectingthe presence of wild type or variant (e.g., mutant or polymorphic) MR-1nucleic acids or polypeptides. The detection of mutant MR-1 polypeptidesfinds use in the diagnosis of disease (e.g., PDC).

A. Detection of Variant MR-1 Alleles

In some embodiments, the present invention provides alleles of MR-1 thatincrease a patient's susceptibility to episodic movement disorders(e.g., PDC). Any mutation that results in an altered phenotype (e.g.,attacks of involuntary movements caused by a neurological dysfunction(e.g., dystonia, chorea, athetosis) lasting up to several hours andoccuring at rest both spontaneously and following caffeine or alcoholconsumption) is within the scope of the present invention.

Accordingly, the present invention provides methods for determiningwhether a patient has an increased susceptibility to a episodic movementdisorder (e.g., PDC) by determining, directly or indirectly, whether theindividual has a variant MR-1 allele. In other embodiments, the presentinvention provides methods for providing a prognosis of increased riskfor episodic movement disorder (e.g., PDC) to an individual based on thepresence or absence of one or more variant alleles of MR-1.

A number of methods are available for analysis of variant (e.g., mutantor polymorphic) nucleic acid or polypeptide sequences. Assays fordetection variants (e.g., polymorphisms or mutations) via nucleic acidanalysis fall into several categories including, but not limited to,direct sequencing assays, fragment polymorphism assays, hybridizationassays, and computer based data analysis. Protocols and commerciallyavailable kits or services for performing multiple variations of theseassays are available. In some embodiments, assays are performed incombination or in hybrid (e.g., different reagents or technologies fromseveral assays are combined to yield one assay). The following exemplaryassays are useful in the present invention: directs sequencing assays,PCR assays, mutational analysis by dHPLC (e.g., available fromTransgenomic, Omaha, Nebr. or Varian, Palo Alto, Calif.), fragmentlength polymorphism assays (e.g., RFLP or CFLP (See e.g. U.S. Pat. Nos.5,843,654; 5,843,669; 5,719,208; and 5,888,780; each of which is hereinincorporated by reference)), hybridization assays (e.g., directdetection of hybridization, detection of hybridization using DNA chipassays (See e.g., U.S. Pat. Nos. 6,045,996; 5,925,525; 5,858,659;6,017,696; 6,068,818; 6,051,380; 6,001,311; 5,985,551; 5,474,796; PCTPublications WO 99/67641 and WO 00/39587, each of which is hereinincorporated by reference), enzymatic detection of hybridization (Seee.g., U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557;5,994,069; 5,962,233; 5,538,848; 5,952,174 and 5,919,626, each of whichis herein incorporated by reference)), polymorphisms detected directlyor indirectly (e.g., detecting sequences (other polymorphisms) that arein linkage disequilibrium with the polymorphism to be indentified; forexample, other sequences in the 2q33-2q35 locus may be used; this methodis described in U.S. Pat. No.: 5,612,179 (herein incorporated byreference)) and mass spectrometry assays.

In addition, assays for the detection of variant MR-1 proteins find usein the present invention (e.g., cell free translation methods, See e.g.,U.S. Pat. No. 6,303,337, herein incorporated by reference) and antibodybinding assays. The generation of antibodies that specifically recognizemutant versus wild type proteins are discussed below.

B. Kits for Analyzing Risk of Episodic Movement Disorders Such As PDC

The present invention also provides kits for determining whether anindividual contains a wild-type or variant (e.g., mutant or polymorphic)allele or polypeptide of MR-1. In some embodiments, the kits are usefuldetermining whether the subject is at risk of developing a episodicmovement disorder (e.g., PDC). The diagnostic kits are produced in avariety of ways. In some embodiments, the kits contain at least onereagent for specifically detecting a mutant MR-1 allele or protein. Inpreferred embodiments, the reagent is a nucleic acid that hybridizes tonucleic acids containing the mutation and that does not bind to nucleicacids that do not contain the mutation. In other embodiments, thereagents are primers for amplifying the region of DNA containing themutation. In still other embodiments, the reagents are antibodies thatpreferentially bind either the wild-type or mutant MR-1 proteins.

In some embodiments, the kit contains instructions for determiningwhether the subject is at risk for a episodic movement disorder (e.g,PDC). In preferred embodiments, the instructions specify that risk fordeveloping a episodic movement disorder such as PDC is determined bydetecting the presence or absence of a mutant MR-1 allele in thesubject, wherein subjects having an mutant allele are at greater riskfor developing the respective disease.

The presence or absence of a disease-associated mutation in a MR-1 genecan be used to make therapeutic or other medical decisions. For example,couples with a family history of episodic movement disorders such as PDCmay choose to conceive a child via in vitro fertilization andpre-implantation genetic screening. In this case, fertilized embryos arescreened for mutant (e.g., disease associated) alleles of a MR-1 geneand only embryos with wild type alleles are implanted in the uterus.

In other embodiments, in utero screening is performed on a developingfetus (e.g., amniocentesis or chorionic villi screening). In still otherembodiments, genetic screening of newborn babies or very young childrenis performed. The early detection of a MR-1 allele known to beassociated with, for example, PDC allows for early intervention (e.g.,genetic or pharmaceutical therapies).

In some embodiments, the kits include ancillary reagents such asbuffering agents, nucleic acid stabilizing reagents, protein stabilizingreagents, and signal producing systems (e.g., florescence generatingsystems as Fret systems). The test kit may be packaged in any suitablemanner, typically with the elements in a single container or variouscontainers as necessary along with a sheet of instructions for carryingout the test. In some embodiments, the kits also preferably include apositive control sample.

C. Bioinformatics

In some embodiments, the present invention provides methods ofdetermining an individual's risk of developing a episodic movementdisorder (e.g., PDC) based on the presence of one or more variantalleles of a MR-1 gene. In some embodiments, the analysis of variantdata is processed by a computer using information stored on a computer(e.g., in a database). For example, in some embodiments, the presentinvention provides a bioinformatics research system comprising aplurality of computers running a multi-platform object orientedprogramming language (See e.g., U.S. Pat. No. 6,125,383; hereinincorporated by reference). In some embodiments, one of the computersstores genetics data (e.g., the risk of developing episodic movementdisorders such as PDC associated with a given polymorphism, as well asthe sequences). In some embodiments, one of the computers storesapplication programs (e.g., for analyzing the results of detectionassays). Results are then delivered to the user (e.g., via one of thecomputers or via the internet.

For example, in some embodiments, a computer-based analysis program isused to translate the raw data generated by the detection assay (e.g.,the presence, absence, or amount of a given MR-1 allele or polypeptide)into data of predictive value for a clinician. The clinician can accessthe predictive data using any suitable means. Thus, in some preferredembodiments, the present invention provides the further benefit that theclinician, who is not likely to be trained in genetics or molecularbiology, need not understand the raw data. The data is presenteddirectly to the clinician in its most useful form. The clinician is thenable to immediately utilize the information in order to optimize thecare of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information providers, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a biopsy or a serum or urine sample) is obtained from asubject and submitted to a profiling service (e.g., clinical lab at amedical facility, genomic profiling business, etc.), located in any partof the world (e.g., in a country different than the country where thesubject resides or where the information is ultimately used) to generateraw data. Where the sample comprises a tissue or other biologicalsample, the subject may visit a medical center to have the sampleobtained and sent to the profiling center, or subjects may collect thesample themselves (e.g., a urine sample) and directly send it to aprofiling center. Where the sample comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., an information card containingthe information may be scanned by a computer and the data transmitted toa computer of the profiling center using an electronic communicationsystems). Once received by the profiling service, the sample isprocessed and a profile is produced (i.e., presence of wild type ormutant MR-1 genes or polypeptides), specific for the diagnostic orprognostic information desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw data, the prepared format may represent a diagnosis orrisk assessment (e.g., likelihood of developing PDC) for the subject,along with recommendations for particular treatment options. The datamay be displayed to the clinician by any suitable method. For example,in some embodiments, the profiling service generates a report that canbe printed for the clinician (e.g., at the point of care) or displayedto the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the association of a given MR-1 allele with an episodicmovement disorder such as PDC.

IV. Generation of MR-1 Antibodies

The present invention provides isolated antibodies or antibody fragments(e.g., FAB fragments). Antibodies can be generated to allow for thedetection of MR-1 proteins (e.g., wild type or mutant) of the presentinvention. The antibodies may be prepared using various immunogens. Inone embodiment, the immunogen is a human MR-1 peptide to generateantibodies that recognize human MR-1. Such antibodies include, but arenot limited to polyclonal, monoclonal, chimeric, single chain, Fabfragments, Fab expression libraries, or recombinant (e.g., chimeric,humanized, etc.) antibodies, as long as it can recognize the protein.Antibodies can be produced by using a protein of the present inventionas the antigen according to a conventional antibody or antiserumpreparation process.

Various procedures known in the art may be used for the production ofpolyclonal antibodies directed against a MR-1 polypeptide. For theproduction of antibody, various host animals can be immunized byinjection with the peptide corresponding to the MR-1 epitope includingbut not limited to rabbits, mice, rats, sheep, goats, etc. In apreferred embodiment, the peptide is conjugated to an immunogeniccarrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyholelimpet hemocyanin (KLH)). Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacille Calmette-Guerin) and Corynebacterium parvum).

For preparation of monoclonal antibodies directed toward MR-1, it iscontemplated that any technique that provides for the production ofantibody molecules by continuous cell lines in culture will find usewith the present invention (See e.g., Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). These include but are not limited to the hybridomatechnique originally developed by Köhler and Milstein (Köhler andMilstein, Nature 256:495-497 [1975]), as well as the trioma technique,the human B-cell hybridoma technique (See e.g., Kozbor et al., Immunol.Tod., 4:72 [1983]), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96 [1985]).

In an additional embodiment of the invention, monoclonal antibodies areproduced in germ-free animals utilizing technology such as thatdescribed in PCT/US90/02545). Furthermore, it is contemplated that humanantibodies will be generated by human hybridomas (Cote et al., Proc.Natl. Acad. Sci. USA 80:2026-2030 [1983]) or by transforming human Bcells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, pp. 77-96 [1985]).

In addition, it is contemplated that techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; hereinincorporated by reference) will find use in producing MR-1 specificsingle chain antibodies. An additional embodiment of the inventionutilizes the techniques described for the construction of Fab expressionlibraries (Huse et al., Science 246:1275-1281 [1989]) to allow rapid andeasy identification of monoclonal Fab fragments with the desiredspecificity for a MR-1 polypeptide.

In other embodiments, the present invention contemplated recombinantantibodies or fragments thereof to the proteins of the presentinvention. Recombinant antibodies include, but are not limited to,humanized and chimeric antibodies. Methods for generating recombinantantibodies are known in the art (See e.g., U.S. Pat. Nos. 6,180,370 and6,277,969 and “Monoclonal Antibodies” H. Zola, BIOS ScientificPublishers Limited 2000. Springer-Verlag New York, Inc., New York; eachof which is herein incorporated by reference).

It is contemplated that any technique suitable for producing antibodyfragments will find use in generating antibody fragments that containthe idiotype (antigen binding region) of the antibody molecule. Forexample, such fragments include but are not limited to: F(ab′)2 fragmentthat can be produced by pepsin digestion of the antibody molecule; Fab′fragments that can be generated by reducing the disulfide bridges of theF(ab′)2 fragment, and Fab fragments that can be generated by treatingthe antibody molecule with papain and a reducing agent.

In the production of antibodies, it is contemplated that screening forthe desired antibody will be accomplished by techniques known in the art(e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),“sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitation reactions, immudiffusion assays, in situ immunoassays(e.g., using colloidal gold, enzyme or radioisotope labels), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide was conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay.)

The foregoing antibodies can be used in methods known in the artrelating to the localization and structure of MR-1 (e.g., for Westernblotting), measuring levels thereof in appropriate biological samples,etc. The antibodies can be used to detect a MR-1 in a biological samplefrom an individual. The biological sample can be a biological fluid,such as, but not limited to, blood, serum, plasma, interstitial fluid,urine, cerebrospinal fluid, and the like, containing cells.

The biological samples can then be tested directly for the presence of ahuman MR-1 using an appropriate strategy (e.g., ELISA orradioimmunoassay) and format (e.g., microwells, dipstick (e.g., asdescribed in International Patent Publication WO 93/03367), etc.Alternatively, proteins in the sample can be size separated (e.g., bypolyacrylamide gel electrophoresis (PAGE), in the presence or not ofsodium dodecyl sulfate (SDS), and the presence of MR-1 detected byimmunoblotting (Western blotting). immunoblotting techniques aregenerally more effective with antibodies generated against a peptidecorresponding to an epitope of a protein, and hence, are particularlysuited to the present invention.

Another method uses antibodies as agents to alter signal transduction.Specific antibodies that bind to the binding domains of MR-1 or otherproteins involved in intracellular signaling can be used to inhibit theinteraction between the various proteins and their interaction withother ligands. Antibodies that bind to the complex can also be usedtherapeutically to inhibit interactions of the protein complex in thesignal transduction pathways leading to the various physiological andcellular effects of MR-1. Such antibodies can also be useddiagnostically to measure abnormal expression of MR-1, or the aberrantformation of protein complexes, which may be indicative of a diseasestate.

V. Gene Therapy Using MR-1

The present invention also provides methods and compositions suitablefor gene therapy to alter MR-1 expression, production, or function. Asdescribed above, the present invention provides human MR-1 genes andprovides methods of obtaining MR-1 genes from other species. Thus, themethods described below are generally applicable across many species. Insome embodiments, it is contemplated that the gene therapy is performedby providing a subject with a wild-type allele of a MR-1 gene (i.e., anallele that does not contain a MR-1 disease allele (e.g., free ofdisease causing polymorphisms or mutations)). Subjects in need of suchtherapy are identified by the methods described above. In someembodiments, transient or stable therapeutic nucleic acids are used(e.g., antisense oligonucleotides, siRNAs) to reduce or preventexpression of mutant proteins.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art (See e.g.,Miller and Rosman, BioTech., 7:980-990 [1992]). Preferably, the viralvectors are replication defective, that is, they are unable to replicateautonomously in the target cell. In general, the genome of thereplication defective viral vectors that are used within the scope ofthe present invention lack at least one region that is necessary for thereplication of the virus in the infected cell. These regions can eitherbe eliminated (in whole or in part), or be rendered non-functional byany technique known to a person skilled in the art. These techniquesinclude the total removal, substitution (by other sequences, inparticular by the inserted nucleic acid), partial deletion or additionof one or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (i.e., on the isolated DNA) or insitu, using the techniques of genetic manipulation or by treatment withmutagenic agents.

Preferably, the replication defective virus retains the sequences of itsgenome that are necessary for encapsidating the viral particles. DNAviral vectors include an attenuated or defective DNA viruses, including,but not limited to, herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, that entirely or almost entirely lack viralgenes, are preferred, as defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt etal., Mol. Cell. Neurosci., 2:320-330 [1991]), defective herpes virusvector lacking a glycoprotein L gene (See e.g., Patent Publication RD371005 A), or other defective herpes virus vectors (See e.g., WO94/21807; and WO 92/05263); an attenuated adenovirus vector, such as thevector described by Stratford-Perricaudet et al. (J. Clin. Invest.,90:626-630 [1992]; See also, La Salle et al., Science 259:988-990[1993]); and a defective adeno-associated virus vector (Samulski et al.,J. Virol., 61:3096-3101 [1987]; Samulski et al., J. Virol., 63:3822-3828[1989]; and Lebkowski et al., Mol. Cell. Biol., 8:3988-3996 [1988]).

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector (e.g.,adenovirus vector), to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-gamma (IFN-γ), or anti-CD4 antibody,can be administered to block humoral or cellular immune responses to theviral vectors. In addition, it is advantageous to employ a viral vectorthat is engineered to express a minimal number of antigens.

In a preferred embodiment, the vector is an adenovirus vector.Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Various serotypes of adenovirus exist. Of these serotypes,preference is given, within the scope of the present invention, to type2 or type 5 human adenoviruses (Ad 2 or Ad 5), or adenoviruses of animalorigin (See e.g., WO 94/26914). Those adenoviruses of animal origin thatcan be used within the scope of the present invention includeadenoviruses of canine, bovine, murine (e.g., Mav1, Beard et al.,Virol., 75-81 [1990]), ovine, porcine, avian, and simian (e.g., SAV)origin. Preferably, the adenovirus of animal origin is a canineadenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61strain (ATCC VR-800)).

Preferably, the replication defective adenoviral vectors of theinvention comprise the ITRs, an encapsidation sequence and the nucleicacid of interest. Still more preferably, at least the E1 region of theadenoviral vector is non-functional. The deletion in the E1 regionpreferably extends from nucleotides 455 to 3329 in the sequence of theAd5 adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfIl-Sau3Afragment). Other regions may also be modified, in particular the E3region (e.g., WO 95/02697), the E2 region (e.g., WO 94/28938), the E4region (e.g., WO 94/28152, WO 94/12649 and WO 95/02697), or in any ofthe late genes L1-L5.

In a preferred embodiment, the adenoviral vector has a deletion in theE1 region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed inEP 185,573, the contents of which are incorporated herein by reference.In another preferred embodiment, the adenoviral vector has a deletion inthe E1 and E4 regions (Ad 3.0). Examples of E1/E4-deleted adenovirusesare disclosed in WO 95/02697 and WO 96/22378. In still another preferredembodiment, the adenoviral vector has a deletion in the E1 region intowhich the E4 region and the nucleic acid sequence are inserted.

The replication defective recombinant adenoviruses according to theinvention can be prepared by any technique known to the person skilledin the art (See e.g., Levrero et al., Gene 101:195 [1991]; EP 185 573;and Graham, EMBO J., 3:2917 [1984]). In particular, they can be preparedby homologous recombination between an adenovirus and a plasmid thatcarries, inter alia, the DNA sequence of interest. The homologousrecombination is accomplished following co-transfection of theadenovirus and plasmid into an appropriate cell line. The cell line thatis employed should preferably (i) be transformable by the elements to beused, and (ii) contain the sequences that are able to complement thepart of the genome of the replication defective adenovirus, preferablyin integrated form in order to avoid the risks of recombination.Examples of cell lines that may be used are the human embryonic kidneycell line 293 (Graham et al., J. Gen. Virol., 36:59 [1977]), whichcontains the left-hand portion of the genome of an Ad5 adenovirus (12%)integrated into its genome, and cell lines that are able to complementthe E1 and E4 functions, as described in applications WO 94/26914 and WO95/02697. Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques that are well known to one ofordinary skill in the art.

The adeno-associated viruses (AAV) are DNA viruses of relatively smallsize that can integrate, in a stable and site-specific manner, into thegenome of the cells that they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The AAV genome has been cloned, sequenced andcharacterized. It encompasses approximately 4700 bases and contains aninverted terminal repeat (ITR) region of approximately 145 bases at eachend, which serves as an origin of replication for the virus. Theremainder of the genome is divided into two essential regions that carrythe encapsidation functions: the left-hand part of the genome, thatcontains the rep gene involved in viral replication and expression ofthe viral genes; and the right-hand part of the genome, that containsthe cap gene encoding the capsid proteins of the virus.

The use of vectors derived from the AAVs for transferring genes in vitroand in vivo has been described (See e.g., WO 91/18088; WO 93/09239; U.S.Pat. No. 4,797,368; U.S. Pat. No. 5,139,941; and EP 488 528, all ofwhich are herein incorporated by reference). These publications describevarious AAV-derived constructs in which the rep and/or cap genes aredeleted and replaced by a gene of interest, and the use of theseconstructs for transferring the gene of interest in vitro (into culturedcells) or in vivo (directly into an organism). The replication defectiverecombinant AAVs according to the invention can be prepared byco-transfecting a plasmid containing the nucleic acid sequence ofinterest flanked by two AAV inverted terminal repeat (ITR) regions, anda plasmid carrying the AAV encapsidation genes (rep and cap genes), intoa cell line that is infected with a human helper virus (for example anadenovirus). The AAV recombinants that are produced are then purified bystandard techniques.

In another embodiment, the gene can be introduced in a retroviral vector(e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289and 5,124,263; all of which are herein incorporated by reference; Mannet al., Cell 33:153 [1983]; Markowitz et al., J. Virol., 62:1120 [1988];PCT/US95/14575; EP 453242; EP178220; Bernstein et al. Genet. Eng., 7:235[1985]; McCormick, BioTechnol., 3:689 [1985]; WO 95/07358; and Kuo etal., Blood 82:845 [1993]). The retroviruses are integrating viruses thatinfect dividing cells. The retrovirus genome includes two LTRs, anencapsidation sequence and three coding regions (gag, pol and env). Inrecombinant retroviral vectors, the gag, pol and env genes are generallydeleted, in whole or in part, and replaced with a heterologous nucleicacid sequence of interest. These vectors can be constructed fromdifferent types of retrovirus, such as, HIV, MoMuLV (“murine Moloneyleukemia virus” MSV (“murine Moloney sarcoma virus”), HaSV (“Harveysarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcomavirus”) and Friend virus. Defective retroviral vectors are alsodisclosed in WO 95/02697.

In general, in order to construct recombinant retroviruses containing anucleic acid sequence, a plasmid is constructed that contains the LTRs,the encapsidation sequence and the coding sequence. This construct isused to transfect a packaging cell line, which cell line is able tosupply in trans the retroviral functions that are deficient in theplasmid. In general, the packaging cell lines are thus able to expressthe gag, pol and env genes. Such packaging cell lines have beendescribed in the prior art, in particular the cell line PA317 (U.S. Pat.No. 4,861,719, herein incorporated by reference), the PsiCRIP cell line(See, WO90/02806), and the GP+envAm-12 cell line (See, WO89/07150). Inaddition, the recombinant retroviral vectors can contain modificationswithin the LTRs for suppressing transcriptional activity as well asextensive encapsidation sequences that may include a part of the gaggene (Bender et al., J. Virol., 61:1639 [1987]). Recombinant retroviralvectors are purified by standard techniques known to those havingordinary skill in the art.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker (Felgneret. al., Proc. Natl. Acad. Sci. USA 84:7413-7417 [1987]; See also,Mackey, et al., Proc. Natl. Acad. Sci. USA 85:8027-8031 [1988]; Ulmer etal., Science 259:1745-1748 [1993]). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Felgner andRingold, Science 337:387-388 [1989]). Particularly useful lipidcompounds and compositions for transfer of nucleic acids are describedin WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127, hereinincorporated by reference.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Methods for formulating and administering naked DNA tomammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and5,589,466, both of which are herein incorporated by reference.

DNA vectors for gene therapy can be introduced into the desired hostcells by methods known in the art, including but not limited totransfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter (See e.g., Wu et al., J. Biol.Chem., 267:963 [1992]; Wu and Wu, J. Biol. Chem., 263:14621 [1988]; andWilliams et al., Proc. Natl. Acad. Sci. USA 88:2726 [1991]).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., Hum. Gene Ther., 3:147 [1992]; and Wu and Wu, J. Biol. Chem.,262:4429 [1987]).

VI. Transgenic Animals Expressing Exogenous MR-1 Genes and Homologs,Mutants, and Variants Thereof

The present invention contemplates the generation of transgenic animalscomprising an exogenous MR-1 gene or homologs, mutants, or variantsthereof. In preferred embodiments, the transgenic animal displays analtered phenotype as compared to wild-type animals. In some embodiments,the altered phenotype is the overexpression of mRNA for a MR-1 gene ascompared to wild-type levels of MR-1 expression. In other embodiments,the altered phenotype is the decreased expression of mRNA for anendogenous MR-1 gene as compared to wild-type levels of endogenous MR-1expression. In some preferred embodiments, the transgenic animalscomprise mutant alleles of MR-1. Methods for analyzing the presence orabsence of such phenotypes include Northern blotting, mRNA protectionassays, and RT-PCR. In other embodiments, the transgenic mice have aknock out mutation of a MR-1 gene. In preferred embodiments, thetransgenic animals display an altered susceptibility to episodicmovement disorders (e.g., PDC).

Such animals find use in research applications (e.g., identifyingsignaling pathways that a MR-1 protein is involved in), as well as drugscreening applications (e.g., to screen for drugs that prevent or treatepisodic movement disorders such as PDC). For example, in someembodiments, test compounds (e.g., a drug that is suspected of beinguseful to treat PDC) are administered to the transgenic animalsexpressing a mutant form of MR-1 and compared with control animalsexpressing a wild type MR-1 allele and the effects evaluated. Theeffects of the test and control compounds on disease symptoms are thenassessed.

The transgenic animals can be generated via a variety of methods. Insome embodiments, embryonal cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonal cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter, which allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. U.S. Pat. No.4,873,191 describes a method for the micro-injection of zygotes; thedisclosure of this patent is incorporated herein in its entirety.

In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is utilized to transfect oocytes by injecting the retroviralvector into the perivitelline space of the oocyte (U.S. Pat. No.6,080,912, incorporated herein by reference). In other embodiments, thedeveloping non-human embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al., in Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1986]). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al., Proc. Natl. Acad. Sci. USA 82:6927 [1985]).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Van der Putten,supra; Stewart, et al., EMBO J., 6:383 [1987]). Alternatively, infectioncan be performed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (Jahner et al., Nature 298:623 [1982]).Most of the founders will be mosaic for the transgene sinceincorporation occurs only in a subset of cells that form the transgenicanimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome that generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germline, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jahner etal., supra [1982]). Additional means of using retroviruses or retroviralvectors to create transgenic animals known to the art involves themicro-injection of retroviral particles or mitomycin C-treated cellsproducing retrovirus into the perivitelline space of fertilized eggs orearly embryos (PCT International Application WO 90/08832 [1990], andHaskell and Bowen, Mol. Reprod. Dev., 40:386 [1995]).

In other embodiments, the transgene is introduced into embryonic stemcells and the transfected stem cells are utilized to form an embryo. EScells are obtained by culturing pre-implantation embryos in vitro underappropriate conditions (Evans et al., Nature 292:154 [1981]; Bradley etal., Nature 309:255 [1984]; Gossler et al., Proc. Acad. Sci. USA 83:9065[1986]; and Robertson et al., Nature 322:445 [1986]). Transgenes can beefficiently introduced into the ES cells by DNA transfection by avariety of methods known to the art including calcium phosphateco-precipitation, protoplast or spheroplast fusion, lipofection andDEAE-dextran-mediated transfection. Transgenes may also be introducedinto ES cells by retrovirus-mediated transduction or by micro-injection.Such transfected ES cells can thereafter colonize an embryo followingtheir introduction into the blastocoel of a blastocyst-stage embryo andcontribute to the germ line of the resulting chimeric animal (forreview, See, Jaenisch, Science 240:1468 [1988]). Prior to theintroduction of transfected ES cells into the blastocoel, thetransfected ES cells may be subjected to various selection protocols toenrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells that haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoel.

In still other embodiments, homologous recombination is utilized toknock-out gene function or create deletion mutants (e.g., mutants inwhich a particular domain of a MR-1 is deleted). Methods for homologousrecombination are described in U.S. Pat. No. 5,614,396, incorporatedherein by reference.

VII. Drug Screening Using MR-1

In some embodiments, the isolated nucleic acid and polypeptides of MR-1genes of the present invention (e.g., SEQ ID NOS: 1, 2, 3 and 4) andrelated proteins and nucleic acids are used in drug screeningapplications for compounds that alter (e.g., enhance or inhibit) MR-1activity and signaling. The present invention further provides methodsof identifying ligands and signaling pathways of the MR-1 proteins ofthe present invention.

The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, based upon the observation that 13 otherepisodic movement disorders are due to ion channel gene mutations (see,e.g., Ptacek L. J., Semin. Neurol., 1999, 19:363-369; hereinincorporated by reference in its entirety), it is contemplated thatmutations within MR-1 results in abnormal ion localization resulting inan episodic movement disorder such as PDC.

In some embodiments, the present invention provides methods of screeningcompounds for the ability to alter MR-1 activity mediated by naturalligands (e.g., identified using the methods described above). Suchcompounds find use in the treatment of disease mediated by MR-1 familymembers (e.g., PDC).

In some embodiments, the present invention provides methods of screeningcompounds for an ability to interact with mutant MR-1 nucleic acid(e.g., SEQ ID NOs: 2, 3 and 4) and/or mutant MR-1 polypeptides (e.g.,SEQ ID NOs: 6, 7 and 8), while simultaneously not interacting with wildtype MR-1 nucleic acid (e.g., SEQ ID NO: 1) and/or wild type MR-1polypeptides (e.g., SEQ ID NO: 5). Such compounds find use in thetreatment of episodic movement disorders facilitated by the presence ofmutant forms of MR-1 nucleic acids and/or proteins.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to MR-1 peptides and isdescribed in detail in WO 84/03564, incorporated herein by reference.Briefly, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are then reacted with MR-1 peptidesand washed. Bound MR-1 peptides are then detected by methods well knownin the art.

Another technique uses MR-1 antibodies, generated as discussed above.Such antibodies are capable of specifically binding to MR-1 peptides andcompete with a test compound for binding to MR-1. In this manner, theantibodies can be used to detect the presence of any peptide that sharesone or more antigenic determinants of a MR-1 peptide.

The present invention contemplates many other means of screeningcompounds. The examples provided above are presented merely toillustrate a range of techniques available. One of ordinary skill in theart will appreciate that many other screening methods can be used.

In particular, the present invention contemplates the use of cell linestransfected with MR-1 genes and variants thereof for screening compoundsfor activity, and in particular to high throughput screening ofcompounds from combinatorial libraries (e.g., libraries containinggreater than 10⁴ compounds). The cell lines of the present invention canbe used in a variety of screening methods. In some embodiments, thecells can be used in second messenger assays that monitor signaltransduction following activation of cell-surface receptors. In otherembodiments, the cells can be used in reporter gene assays that monitorcellular responses at the transcription/translation level. In stillfurther embodiments, the cells can be used in cell proliferation assaysto monitor the overall growth/no growth response of cells to externalstimuli.

In second messenger assays, the host cells are preferably transfected asdescribed above with vectors encoding MR-1 or variants or mutantsthereof. The host cells are then treated with a compound or plurality ofcompounds (e.g., from a combinatorial library) and assayed for thepresence or absence of a response. It is contemplated that at least someof the compounds in the combinatorial library can serve as agonists,antagonists, activators, or inhibitors of the protein or proteinsencoded by the vectors. It is also contemplated that at least some ofthe compounds in the combinatorial library can serve as agonists,antagonists, activators, or inhibitors of protein acting upstream ordownstream of the protein encoded by the vector in a signal transductionpathway.

In some embodiments, the second messenger assays measure fluorescentsignals from reporter molecules that respond to intracellular changes(e.g., Ca²⁺ concentration, membrane potential, pH, IP₃, cAMP,arachidonic acid release) due to stimulation of membrane receptors andion channels (e.g., ligand gated ion channels; see Denyer et al., DrugDiscov. Today 3:323 [1998]; and Gonzales et al., Drug. Discov. Today4:431-39 [1999]). Examples of reporter molecules include, but are notlimited to, FRET (florescence resonance energy transfer) systems (e.g.,Cuo-lipids and oxonols, EDAN/DABCYL), calcium sensitive indicators(e.g., Fluo-3, FURA 2, INDO 1, and FLUO3/AM, BAPTA AM),chloride-sensitive indicators (e.g. SPQ, SPA), potassium-sensitiveindicators (e.g., PBFI), sodium-sensitive indicators (e.g., SBFI), andpH sensitive indicators (e.g., BCECF).

In general, the host cells are loaded with the indicator prior toexposure to the compound. Responses of the host cells to treatment withthe compounds can be detected by methods known in the art, including,but not limited to, fluorescence microscopy, confocal microscopy (e.g.,FCS systems), flow cytometry, microfluidic devices, FLIPR systems (See,e.g., Schroeder and Neagle, J. Biomol. Screening 1:75 [1996]), andplate-reading systems. In some preferred embodiments, the response(e.g., increase in fluorescent intensity) caused by compound of unknownactivity is compared to the response generated by a known agonist andexpressed as a percentage of the maximal response of the known agonist.The maximum response caused by a known agonist is defined as a 100%response. Likewise, the maximal response recorded after addition of anagonist to a sample containing a known or test antagonist is detectablylower than the 100% response.

The cells are also useful in reporter gene assays. Reporter gene assaysinvolve the use of host cells transfected with vectors encoding anucleic acid comprising transcriptional control elements of a targetgene (i.e., a gene that controls the biological expression and functionof a disease target) spliced to a coding sequence for a reporter gene.Therefore, activation of the target gene results in activation of thereporter gene product. In some embodiments, the reporter gene constructcomprises the 5′ regulatory region (e.g., promoters and/or enhancers) ofa protein whose expression is controlled by MR-1 in operable associationwith a reporter gene. Examples of reporter genes finding use in thepresent invention include, but are not limited to, chloramphenicoltransferase, alkaline phosphatase, firefly and bacterial luciferases,β-galactosidase, β-lactamase, and green fluorescent protein. Theproduction of these proteins, with the exception of green fluorescentprotein, is detected through the use of chemiluminescent, calorimetric,or bioluminecent products of specific substrates (e.g., X-gal andluciferin). Comparisons between compounds of known and unknownactivities may be conducted as described above.

Specifically, the present invention provides screening methods foridentifying modulators, i.e., candidate or test compounds or agents(e.g., proteins, peptides, peptidomimetics, peptoids, small molecules orother drugs) which bind to a MR-1 of the present invention, have aninhibitory (or stimulatory) effect on, for example, MR-1 expression orMR-1 activity, or have a stimulatory or inhibitory effect on, forexample, the expression or activity of a MR-1 substrate. Compounds thusidentified can be used to modulate or replace the activity of targetgene products (e.g., MR-1 genes) either directly or indirectly in atherapeutic protocol, to elaborate the biological function of the targetgene product, or to identify compounds that disrupt normal target geneinteractions. Compounds, which stimulate the activity of a variant MR-1or mimic the activity of a non-functional variant are particularlyuseful in the treatment of episodic movement disorders (e.g., PDC).

In one embodiment, the invention provides assays for screening candidateor test compounds that are substrates of a MR-1 protein or polypeptideor a biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compounds thatbind to or modulate the activity of a MR-1 protein or polypeptide or abiologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone, which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckennann et al., J.Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are preferred for use withpeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909 [1993]; Erb et al., Proc. Natl. Acad. Sci. USA 91:11422[1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al.,Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061[1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84[1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores(U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids(Cull et al., Proc. Natl. Acad. Sci. USA 89:18651869 [1992]) or on phage(Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406[1990]; Cwirla et al., Proc. Natl. Acad. Sci. 87:6378-6382 [1990];Felici, J. Mol. Biol. 222:301 [1991]).

Modulators of MR-1 expression can also be identified. For example, acell or cell free mixture is contacted with a candidate compound and theexpression of a MR-1 mRNA or protein evaluated relative to the level ofexpression of the MR-1 mRNA or protein in the absence of the candidatecompound. When expression of the MR-1 mRNA or protein is greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of a MR-1 mRNA or proteinexpression. Alternatively, when expression of MR-1 mRNA or protein isless (i.e., statistically significantly less) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as an inhibitor of MR-1 mRNA or protein expression. The levelof MR-1 mRNA or protein expression can be determined by methodsdescribed herein for detecting MR-1 mRNA or protein.

A modulating agent can be identified using a cell-based or a cell freeassay, and the ability of the agent to modulate the activity of a MR-1protein can be confirmed in vivo, e.g., in an animal such as an animalmodel for a disease (e.g., an animal with PDC).

B. Therapeutic Agents

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a MR-1 modulating agent or mimetic, a MR-1 specific antibody, ora MR-1 -binding partner) in an appropriate animal model (such as thosedescribed herein) to determine the efficacy, toxicity, side effects, ormechanism of action, of treatment with such an agent. Furthermore, asdescribed above, novel agents identified by the above-describedscreening assays can be, e.g., used for treatments of episodic movementdisorders (e.g., including, but not limited to, PDC). In someembodiments, the agents are MR-1 ligands or ligand analogs (e.g.,identified using the drug screening methods described above).

VIII. Pharmaceutical Compositions Containing MR-1 Nucleic Acid,Peptides, and Analogs

The present invention further provides pharmaceutical compositions whichmay comprise all or portions of MR-1 polynucleotide sequences, MR-1polypeptides, inhibitors or antagonists of MR-1 bioactivity, includingantibodies, alone or in combination with at least one other agent, suchas a stabilizing compound, and may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water.

The methods of the present invention find use in treating diseases oraltering physiological states characterized by mutant MR-1 alleles(e.g., episodic movement disorders such as PDC). Peptides can beadministered to the patient intravenously in a pharmaceuticallyacceptable carrier such as physiological saline. Standard methods forintracellular delivery of peptides can be used (e.g., delivery vialiposome). Such methods are well known to those of ordinary skill in theart. The formulations of this invention are useful for parenteraladministration, such as intravenous, subcutaneous, intramuscular, andintraperitoneal. Therapeutic administration of a polypeptideintracellularly can also be accomplished using gene therapy as describedabove.

As is well known in the medical arts, dosages for any one patientdepends upon many factors, including the patient's size, body surfacearea, age, the particular compound to be administered, sex, time androute of administration, general health, and interaction with otherdrugs being concurrently administered.

Accordingly, in some embodiments of the present invention, MR-1nucleotide and MR-1 amino acid sequences can be administered to apatient alone, or in combination with other nucleotide sequences, drugsor hormones or in pharmaceutical compositions where it is mixed withexcipient(s) or other pharmaceutically acceptable carriers. In oneembodiment of the present invention, the pharmaceutically acceptablecarrier is pharmaceutically inert. In another embodiment of the presentinvention, MR-1 polynucleotide sequences or MR-1 amino acid sequencesmay be administered alone to individuals subject to or suffering from adisease.

Depending on the condition being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; as well as parenteral delivery, includingintramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intraperitoneal, or intranasaladministration.

For injection, the pharmaceutical compositions of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. For tissue or cellular administration,penetrants appropriate to the particular barrier to be permeated areused in the formulation. Such penetrants are generally known in the art.

In other embodiments, the pharmaceutical compositions of the presentinvention can be formulated using pharmaceutically acceptable carrierswell known in the art in dosages suitable for oral administration. Suchcarriers enable the pharmaceutical compositions to be formulated astablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like, for oral or nasal ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. For example, aneffective amount of MR-1 may be that amount that suppresses PDC relatedsymptoms (e.g., involuntary movement). Determination of effectiveamounts is well within the capability of those skilled in the art,especially in light of the disclosure provided herein.

In addition to the active ingredients these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of the activecompounds into preparations that can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes).

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are carbohydrate or protein fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; starch from corn,wheat, rice, potato, etc; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, (i.e., dosage).

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients mixed with a filler orbinders such as lactose or starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

Compositions comprising a compound of the invention formulated in apharmaceutical acceptable carrier may be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. For polynucleotide or amino acid sequences of MR-1,conditions indicated on the label may include treatment of conditionrelated to episodic movement disorders such as PDC.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. Then, preferably, dosage can be formulated in animalmodels (particularly murine models) to achieve a desirable circulatingconcentration range that adjusts MR-1 levels.

A therapeutically effective dose refers to that amount of MR-1 thatameliorates symptoms of the disease state. Toxicity and therapeuticefficacy of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds thatexhibit large therapeutic indices are preferred. The data obtained fromthese cell culture assays and additional animal studies can be used informulating a range of dosage for human use. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage varieswithin this range depending upon the dosage form employed, sensitivityof the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state; age, weight, and gender of the patient;diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy. Long actingpharmaceutical compositions might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular formulation.

Normal dosage amounts may vary from 0.01 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature (See, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212,all of which are herein incorporated by reference). Those skilled in theart will employ different formulations for MR-1 than for the inhibitorsof MR-1. Administration to the bone marrow may necessitate delivery in amanner different from intravenous injections.

IX. RNAi for MR-1

The present invention provides RNAi for inhibiting the expression of theMR-1 polypeptide in cells for research or therapeutic applications.

A. Designing and Testing RNAi for MR-1

In order to design siRNAs for MR-1 (e.g. that target MR-1 mRNA) softwaredesign tools are available in the art. For example, Oligoengine's webpage has one such design tool that finds RNAi candidates based onElbashir's (Elbashir et al, Methods 2002; 26: 199-213, hereinincorporated by reference) criteria. Other design tools may also beused, such as the Cenix Bioscience design tool offered by Ambion. Inaddition, there is also the Si2 silencing duplex offered by Oligoengine.

There are also RNA folding software programs available that allow one todetermine if the mRNA has a tendency to fold on its own and form a“hair-pin” (which in the case of dsRNAi is not as desirable since onegoal is to have the RNAi attach to the mRNA and not itself). Onepreferred configuration is an open configuration with three or lessbonds. Generally, a positive delta G is desirable to show that it wouldnot tend to fold on itself spontaneously.

siRNA candidate molecules that are generated can be, for example,screened in an animal model of PDC for the quantitative evaluation ofMR-1 expression in vivo using similar techniques as described above.

B. Expression Cassettes

MR-1 specific siRNAs of the present invention may be synthesizedchemically. Chemical synthesis can be achieved by any method known ordiscovered in the art. Alternatively, MR-1 specific siRNAs of thepresent invention may be synthesized by methods which comprise synthesisby transcription. In some embodiments, transcription is in vitro, asfrom a DNA template and bacteriophage RNA polymerase promoter, in otherembodiments, synthesis is in vivo, as from a gene and a promoter.Separate-stranded duplex siRNA, where the two strands are synthesizedseparately and annealed, can also be synthesized chemically by anymethod known or discovered in the art. Alternatively, ds siRNA aresynthesized by methods which comprise synthesis by transcription. Insome embodiments, the two strands of the double-stranded region of asiRNA are expressed separately by two different expression cassettes,either in vitro (e.g., in a transcription system) or in vivo in a hostcell, and then brought together to form a duplex.

Thus, in another aspect, the present invention provides a compositioncomprising an expression cassette comprising a promoter and a gene thatencodes a siRNA specific for MR-1. In some embodiments, the transcribedsiRNA forms a single strand of a separate-stranded duplex (ordouble-stranded, or ds) siRNA of about 18 to 25 base pairs long; thus,formation of ds siRNA requires transcription of each of the twodifferent strands of a ds siRNA. The term “gene” in the expressioncassette refers to a nucleic acid sequence that comprises codingsequences necessary for the production of a siRNA. Thus, a gene includesbut is not limited to coding sequences for a strand of a ds siRNA.

Generally, a DNA expression cassette comprises a chemically synthesizedor recombinant DNA molecule containing at least one gene, or desiredcoding sequence for a single strand of a ds siRNA, and appropriatenucleic acid sequences necessary for the expression of the operablylinked coding sequence, either in vitro or in vivo. Expression in vitromay include expression in transcription systems and intranscription/translation systems. Expression in vivo may includeexpression in a particular host cell and/or organism. Nucleic acidsequences necessary for expression in a prokaryotic cell or in aprokaryotic in vitro expression system are well known and usuallyinclude a promoter, an operator, and a ribosome binding site, oftenalong with other sequences. Eukaryotic in vitro transcription systemsand cells are known to utilize promoters, enhancers, and termination andpolyadenylation signals. Nucleic acid sequences necessary for expressionvia bacterial RNA polymerases (such as T3, T7, and SP6), referred to asa transcription template in the art, include a template DNA strand whichhas a polymerase promoter region followed by the complement of the RNAsequence desired (or the coding sequence or gene for the siRNA). Inorder to create a transcription template, a complementary strand isannealed to the promoter portion of the template strand.

In any of the expression cassettes described above, the gene may encodea transcript that contains at least one cleavage site, such that whencleaved results in at least two cleavage products. Such products caninclude the two opposite strands of a ds siRNA. In an expression systemfor expression in a eukaryotic cell, the promoter may be constitutive orinducible; the promoter may also be tissue or organ specific (e.g.specific to the eye), or specific to a developmental phase. Preferably,the promoter is positioned 5′ to the transcribed region. Other promotersare also contemplated; such promoters include other polymerase IIIpromoters and microRNA promoters.

Preferably, a eukaryotic expression cassette further comprises atranscription termination signal suitable for use with the promoter; forexample, when the promoter is recognized by RNA polymerase III, thetermination signal is an RNA polymerase III termination signal. Thecassette may also include sites for stable integration into a host cellgenome.

C. Vectors

In other aspects of the present invention, the compositions comprise avector comprising a gene encoding an siRNA specific for MR-1 orpreferably at least one expression cassette comprising a promoter and agene which encodes a sequence necessary for the production of a siRNAspecific for MR-1 (an siRNA gene). The vectors may further comprisemarker genes, reporter genes, selection genes, or genes of interest,such as experimental genes. Vectors of the present invention includecloning vectors and expression vectors. Expression vectors may be usedin in vitro transcription/translation systems, as well as in in vivo ina host cell. Expression vectors used in vivo in a host cell may betransfected into a host cell, either transiently, or stably. Thus, avector may also include sites for stable integration into a host cellgenome.

In some embodiments, it is useful to clone a siRNA gene downstream of abacteriophage RNA polymerase promoter into a multicopy plasmid. Avariety of transcription vectors containing bacteriophage RNA polymerasepromoters (such as T7 promoters) are available. Alternatively, DNAsynthesis can be used to add a bacteriophage RNA polymerase promoterupstream of a siRNA coding sequence. The cloned plasmid DNA, linearizedwith a restriction enzyme, can then be used as a transcription template(See for example Milligan, J F and Uhlenbeck, O C (1989) Methods inEnzymology 180: 51-64).

In other embodiments of the present invention, vectors include, but arenot limited to, chromosomal, nonchromosomal and synthetic DNA sequences(e.g., derivatives of viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies). It is contemplated that any vector may be usedas long as it is expressed in the appropriate system (either in vitro orin vivo) and viable in the host when used in vivo; these two criteriaare sufficient for transient transfection. For stable transfection, thevector is also replicable in the host.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. In some embodiments of the presentinvention, mammalian expression vectors comprise an origin ofreplication, suitable promoters and enhancers, and also any necessaryribosome binding sites, polyadenylation sites, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnon-transcribed sequences. In other embodiments, DNA sequences derivedfrom the SV40 splice, and polyadenylation sites may be used to providethe required non-transcribed genetic elements.

In certain embodiments of the present invention, a gene sequence in anexpression vector which is not part of an expression cassette comprisinga siRNA gene (specific for MR-1) is operatively linked to an appropriateexpression control sequence(s) (promoter) to direct mRNA synthesis. Insome embodiments, the gene sequence is a marker gene or a selectiongene. Promoters useful in the present invention include, but are notlimited to, the cytomegalovirus (CMV) immediate early, herpes simplexvirus (HSV) thymidine kinase, and mouse metallothionein promoters andother promoters known to control expression of gene in mammalian cellsor their viruses. In other embodiments of the present invention,recombinant expression vectors include origins of replication andselectable markers permitting transformation of the host cell (e.g.,dihydrofolate reductase or neomycin resistance for eukaryotic cellculture).

In some embodiments of the present invention, transcription of DNAencoding a gene is increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp that act on a promoter to increase its transcription.Enhancers useful in the present invention include, but are not limitedto, a cytomegalovirus early promoter enhancer, the polyoma enhancer onthe late side of the replication origin, and adenovirus enhancers.

Preferably the design of a vector is configured to deliver the RNAi formore permanent inhibition. For example the pSilencer siRNA expressionvector offered by Ambion, the pSuper RNAi system offered by Oligoengine,and the GneSilencer System offered by IMGENEX. These are all plasmidvector based RNAis. BD Biosciences offer the RNAi-Ready pSIREN Vectors,that allow both a Plasmid-based vectors and an Adenoviral or aRetroviral delivery formats. Ambion is expected to release an adenoviralvector for siRNA shortly. For the design of a vector there is nolimitation regarding the folding pattern since there is no concernregarding the formation of a hairpin or at least there are no studiesthat found any difference in performance related to the mRNA foldingpattern.

It is noted that Ambion offers a design tool for a vector on their webpage, and BD Biosciences offers a manual for the design of a vector,both of which are useful for designing vectors for siRNA.

D. Transfecting Cells

In yet other aspects, the present invention provides compositionscomprising cells transfected by an expression cassette of the presentinvention as described above, or by a vector of the present invention,where the vector comprises an expression cassette (or simply the siRNAgene) of the present invention, as described above. In some embodimentsof the present invention, the host cell is a mammalian cell. Atransfected cell may be a cultured cell or a tissue, organ, ororganismal cell. Specific examples of cultured host cells include, butare not limited to, Chinese hamster ovary (CHO) cells, COS-7 lines ofmonkey kidney fibroblasts, 293T, C127, 3T3, HeLa, and BHK cell lines.Specific examples of host cells in vivo include tumor tissue and eyetissue.

The cells may be transfected transiently or stably (e.g. DNA expressingthe siRNA is stably integrated and expressed by the host cell's genome).The cells may also be transfected with an expression cassette of thepresent invention, or they are transfected with an expression vector ofthe present invention. In some embodiments, transfected cells arecultured mammalian cells, preferably human cells. In other embodiments,they are tissue, organ, or organismal cells.

In the present invention, cells to be transfected in vitro are typicallycultured prior to transfection according to methods which are well knownin the art, as for example by the preferred methods as defined by theAmerican Tissue Culture Collection. In certain embodiments of thepresent invention, cells are transfected with siRNAs that aresynthesized exogenously (or in vitro, as by chemical methods or in vitrotranscription methods), or they are transfected with expressioncassettes or vectors, which express siRNAs within the transfected cell.

In some embodiments, cells are transfected with siRNAs by any methodknown or discovered in the art which allows a cell to take up exogenousRNA and remain viable. Non-limiting examples include electroporation,microinjection, transduction, cell fusion, DEAE dextran, calciumphosphate precipitation, use of a gene gun, osmotic shock, temperatureshock, and electroporation, and pressure treatment. In alternative,embodiments, the siRNAs are introduced in vivo by lipofection, as hasbeen reported (as, for example, by Elbashir et al. (2001) Nature 411:494-498, herein incorporated by reference).

In other embodiments expression cassettes or vectors comprising at leastone expression cassette are introduced into the desired host cells bymethods known in the art, including but not limited to transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (See e.g., Wu et al. (1992) J. Biol. Chem.,267:963; Wu and Wu (1988) J. Biol. Chem., 263:14621; and Williams et al.(1991) Proc. Natl. Acad. Sci. USA 88:272). Receptor-mediated DNAdelivery approaches are also used (Curiel et al. (1992) Hum. Gene Ther.,3:147; and Wu and Wu (1987) J. Biol. Chem., 262:4429). In someembodiments, various methods are used to enhance transfection of thecells. These methods include but are not limited to osmotic shock,temperature shock, and electroporation, and pressure treatment.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker. The useof cationic lipids may promote encapsulation of negatively chargednucleic acids, and also promote fusion with negatively charged cellmembranes. Particularly useful lipid compounds and compositions fortransfer of nucleic acids are described in WO95/18863 and WO96/17823,and in U.S. Pat. No. 5,459,127, herein incorporated by reference. Othermolecules are also useful for facilitating transfection of a nucleicacid in vivo, such as a cationic oligopeptide (e.g., WO95/21931),peptides derived from DNA binding proteins (e.g., WO96/25508), or acationic polymer (e.g., WO95/21931).

It is also possible to introduce a sequence encoding a siRNA in vivo asa naked DNA, either as an expression cassette or as a vector. Methodsfor formulating and administering naked DNA to mammalian muscle tissueare disclosed in U.S. Pat. Nos. 5,580,859 and 5,589,466, both of whichare herein incorporated by reference.

Stable transfection typically requires the presence of a selectablemarker in the vector used for transfection. Transfected cells are thensubjected to a selection procedure. Generally, selection involvesgrowing the cells in a toxic substance, such as G418 or Hygromycin B,such that only those cells expressing a transfected marker geneconferring resistance to the toxic substance upon the transfected cellsurvive and grow. Such selection techniques are well known in the art.Typical selectable markers are well known, and include genes encodingresistance to G418 or hygromycin B.

In preferred embodiments, the transfecting agent is OLIGOFECTAMINE.OLIGOFECTAMINE is a lipid based transfection reagent. Additional exampleof lipid based transfection reagents that were designed for thetransfection of dsRNAis are the Transit-TKO reagent which is provided byMirus (Madison, Wis.) and the jetSI which was introduced byPolyplus-trasfection SAS. In addition, the Silencer siRNA TransfectionKit provided by Ambion's includes siPORT Amine and siPORT Lipidtransfection agents. Roche offers the Fugene 6 transfection reagentsthat are also lipid based. There is an option to use electroporation incell culture. Preferably a plasmid vector delivery system is transfectedinto the cell with OLIGOFECTAMINE provided by Invitrogen or with siPORTXP-1 transfection agent provided by Ambion.

In certain embodiments, certain chemical modifications of the dsRNAissuch as changing the lipophilicity of the molecule may be employed(e.g., attachment of lipophilic residues at the 3′ termini of thedsRNA). Delivery of dsRNAs into organisms may also be achieved withmethods previously developed for the application of antisenseoligonucleotides such as injection of liposomes-encapsulated molecules.

E. Kits

The present invention also provides kits comprising at least oneexpression cassette comprising a siRNA gene specific for MR-1 or avariant of MR-1. In some aspects, a transcript from the expressioncassette forms a double stranded siRNA of about 18 to 25 base pairslong. In other embodiments, the expression cassette is contained withina vector, as described above, where the vector can be used in in vitrotranscription or transcription/translation systems, or used in vivo totransfect cells, either transiently or stably.

In other aspects, the kit comprises at least two expression cassettes,each of which comprises a siRNA gene, such that at least one geneencodes one strand of a siRNA that combines with a strand encoded by asecond cassette to form a ds siRNA; the ds siRNA so produced is any ofthe embodiments described above. These cassettes may comprise a promoterand a sequence encoding one strand of a ds siRNA. In some furtherembodiments, the two expression cassettes are present in a singlevector; in other embodiments, the two expression cassettes are presentin two different vectors. A vector with at least one expressioncassette, or two different vectors, each comprising a single expressioncassette, can be used in in vitro transcription ortranscription/translation systems, or used in vivo to transfect cells,either transiently or stably.

In yet other aspects, the kit comprises at least one expressioncassettes which comprises a gene which encodes two separate strands of ads siRNA and a processing site between the sequences encoding eachstrand such that, when the gene is transcribed, the transcript isprocessed, such as by cleavage, to result in two separate strands whichcan combine to form a ds siRNA, as described above.

In some embodiments, the present invention provides kits comprising; a)a composition comprising small interfering RNA duplexes (siRNAs)configured to inhibit expression of an MR-1 protein, and/or b) printedmaterial with instructions for employing the composition for treating atarget cell expressing MR-1 protein via expression of MR-1 mRNA underconditions such that the MR-1 mRNA is cleaved or otherwise disabled. Incertain embodiments, the printed material comprises instructions foremploying the composition for treating eye disease.

F. Generating MR-1 Specific siRNA

The present invention also provides methods of synthesizing siRNAsspecific for MR-1 (e.g. human MR-1) or specific for mutant or wild typeforms of MR-1. The siRNAs may be synthesized in vitro or in vivo. Invitro synthesis includes chemical synthesis and synthesis by in vitrotranscription. In vitro transcription is achieved in a transcriptionsystem, as from a bacteriophage RNA polymerase, or in atranscription/translation system, as from a eukaryotic RNA polymerase.In vivo synthesis occurs in a transfected host cell.

The siRNAs synthesized in vitro, either chemically or by transcription,are used to transfect cells. Therefore, the present invention alsoprovides methods of transfecting host cells with siRNAs synthesized invitro; in particular embodiments, the siRNAs are synthesized by in vitrotranscription. The present invention further provides methods ofsilencing the MR-1 gene in vivo by transfecting cells with siRNAssynthesized in vitro. In other methods, the siRNAs is expressed in vitroin a transcription/translation system from an expression cassette orexpression vector, along with an expression vector encoding andexpressing a reporter gene.

The present invention also provides methods of expressing siRNAs in vivoby transfecting cells with expression cassettes or vectors which directsynthesis of siRNAs in vivo. The present invention also provides methodsof silencing genes in vivo by transfecting cells with expressioncassettes or vectors that direct synthesis of siRNAs in vivo.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1

This example describes the subjects used in experiments conducted duringthe course of the present invention. Subjects participated according tothe University of Michigan (Ann Arbor) institutionalreviewboard-approved protocol. Subjects were diagnosed as eitheraffected with PDC or unaffected prior to genetic linkage and candidategene analysis. Diagnosis was based on normal developmental milestones,observered descriptions of episodes consistent with nonkinesigenic,nonhypnogenic paroxysmal dyskinesia lasting longer than 5 minutes,normal interictal neurologic examination results, and witnessed episodesthat were consistent with PDC. Control subjects were older than 60 yearsand were determined by neurological examination and structuredpsychiatric interview to have no personal or family history ofneurologic or psychiatric disorders.

Example 2

This example describes the performed genetic linkage analysis. DNA wasextracted from peripheral blood leukocytes, microsatellite DNApolymorphisms, and 2-point lod scores calculated as previously described(see, e.g., Rainier S., et al., Am J Hum Genet. 2003, 73:967-971; ZhaoX., et al., Nat Genet. 2001, 29:326-331; each herein incorporated byreference in their entireties). Genetic linkage analysis of the PDC-Detkindred (substitution of valine for alanine at amino acid position 9)(see FIG. 9) was reported previously (see, e.g., Fink J. K., et al., AmJ Hum Genet. 1996, 59:140-145; herein incorporated by reference in itsentirety). For the PDC-Pa kindred (substitution of valine for alanine atamino acid position 7) (see, e.g., FIG. 9), genetic linkage between thedisorder and the PDC locus was assessed by the examination of markersD2S295, D2S2210, D2S434, D2S2249, D2S94, D2S173, D2S2179, D2S104,D2S2250, D2S433, D2S2244, D2S2151, and D2S163 using an autosomaldominant mode of inheritance, applying an assumed disease allelefrequency of 0.001, and assigning genetic penetrance to 0.90. Allelefrequencies were assumed to be equal because there were too fewmarrying-in spouses to calculate allele frequencies accurately.

Example 3

This example describes the physical mapping of the PDC locus interval. Aphysical map across the consensus PDC locus interval (D2S295-D2S163)consisting of 22 overlapping bacterial artificial chromosome elementswas created. Subsequently, the Human Genome Project created overlappingcontigs (NT_(—)005337 and NT_(—)005289, that were combined into contigNT_(—)005403 that included the 2.4-Mb PDC locus and for which DNAsequences were made publicly available. It was confirmed that thesequence tagged site (STS) content of these contigs by a combination ofSTS amplification from individual bacterial artificial chromosomeelements and Basic Local Alignment Search Tool analysis to determine ifthe DNA sequences of given STSs were contained in the annotated contigsequence.

Example 4

This example describes the identification, prioritization and analysisof the PDC candidate genes. 116 potential candidate genes wereidentified in the PDC contig by analysis of expressed sequence tags andcomplementary DNA (cDNA) sequences listed with annotated contigs fromthe National Center for Biotechnology Information (Bethesda, Md.) and byPipeline analysis of contig and individual bacterial artificialchromosome DNA sequences. Involuntary movements in PDC involve (but donot necessarily originate in) the extrapyramidal motor system.Therefore, to prioritize the analysis of 116 positional candidate genes,reverse transcription-polymerase chain reaction (RT-PCR) was used todetermine which candidate genes were expressed in the brain. For this, aSuperscript RT-PCR kit (Invitrogen, Carlsbad, Calif.) was used toamplify candidate genes from adult brain messenger RNA (mRNA)(Stratagene, La Jolla, Calif.). Whenever possible, exonic primers wereused to amplify across small introns so that it could be determined byamplification fragment size whether the template consisted of cDNA orcontaminating genomic DNA. When intronic sequences were less than 3kilobases (kb), RT-PCR amplification products was compared with thoseobtained from the genomic DNA template.

Example 5

This example describes an analysis of MR-1 gene expression in multipletissues by RT-PCR. This analysis was performed using a SuperscriptRT-PCR kit (Invitrogen) to amplify a fragment of the MR-1 gene from theadult brain, liver, kidney, skeletal muscle, heart, and lung mRNA(Stratagene). Placement of the forward primer(5_-ATCTGAACATGGCGGCGGTGGTAG-3_) (SEQ ID NO: 9) in MR-1 NM_(—)015488exon 1 and the reverse primer (5_-AGTGGCCTTTAGGGTAGCGATTCC-3_) (SEQ IDNO: 10) in MR-1 NM_(—)015488 exon 3 resulted in a 333-base pair (bp)cDNA amplification product. Inclusion of introns 1 and 2 allowed sizediscrimination of amplification products from mRNA (333 bp) and genomicDNA templates (_(—)50 kb). The RT-PCR amplification of a 626-bp β-actinmRNA fragment served as a control and was performed with previouslydescribed primers and methods (see, e.g., Raff T., et al., Biotechniques1997, 23:456-460; herein incorporated by reference in its entirety). Theconditions were the same for both MR-1 and β-actin amplification (50° C.for 30 minutes, denaturation for 2 minutes at 94° C., followed by 34cycles each at 94° C. for 15 seconds, 55° C. for 30 seconds, and 72° C.for 1 minute, followed by 10-minute elongation at 72° C.).

Example 6

This example describes protein sequence analysis. The MR-1 homologueswere found using Tblastn, the protein query of the translated database.The secondary structure prediction was performed using Protean sequenceanalysis software (DNASTAR, Madison, Wis.).

Example 7

This example describes the clinical features of the subjects. Clinicalfeatures and (+)-α-[11C] dihydrotetrabenazine positron emissiontomography for the PDC-Det kindred have previously been reported (see,e.g., Fink J. K., et al., Neurology. 1997, 49:177-183; Bohnen N. I., etal., Neurology. 1999, 52:1067-1069; each herein incorporated byreference in their entireties). The PDC-Det kindred was of Polishancestry, and the PDC-Pa kindred was of English and mixed Europeanancestry. The clinical syndrome of early childhood-onset nonkinesigenicdyskinesia in affected subjects in the PDC-Pa family was very similar tothat previously described for the PDC-Det family (see, e.g., Fink J. K.,et al., Neurology. 1997, 49:177-183; herein incorporated by reference inits entirety). Affected subjects in the PDC-Pa kindred experiencedepisodes (ranging from once a week to several times a day) ofinvoluntary movements involving the face and all extremities that lastedfrom 5 minutes to more than 1 hour. Episodes occurred spontaneouslywhile at rest and following caffeine or alcohol consumption. Theseinvoluntary movements did not occur during sleep, when falling asleep,or when waking up and were not provoked by exercise or sudden movement.Developmental milestone and interictal neurologic examination resultswere normal in all subjects with the exception of 1 individual who hadchildhood-onset polio and 1 subject who had facial tics (blinking), bothof whom were in the PDC-Det kindred and have been described previously(see, e.g., Fink J. K., et al., Neurology. 1997, 49:177-183; hereinincorporated by reference in its entirety).

Example 8

This example describes the linkage of the disorder in PDC-DET and PDC-PAkindreds to Chromosome 2q33-2q35. Linkage of this disorder to chromosome2q33-2q35 in the PDC-Det kindred (maximum 2-point lod score, +4.77 atθ=0 for marker D2S173 [AFM249 wg9]) has been reported (see, e.g., FinkJ. K., Am J Hum Genet. 1996, 59:140-145; herein incorporated byreference in its entirety). Analysis of the PDC-Pa kindred (FIG. 9) wasalso consistent with linkage to this locus (maximum 2-point lod score,+2.41 at _(—)=0 for D2S163).

Example 9

This example describes haplotype analysis. Extended haplotypes forlinked markers in PDC-Det and PDC-Pa families was analyzed and noevidence of haplotype sharing was found. Therefore, there was noevidence that these 2 families were closely related.

Example 10

This example describes the identification and analysis of PDC positionalcandidate genes. RT-PCR analysis provided evidence that 45 of the 116known and putative genes in the PDC locus interval were transcribed inthe brain. Intron-exon boundaries were identified, and candidate genesequencing was performed as previously described (see, e.g., Rainier S.,et al., Am J Hum Genet. 2003, 73:967-971; Zhao X., et al., Nat Genet.2001; 29:326-331; each herein incorporated by reference in theirentireties). Analysis of 17 of these genes did not disclosePDC-specific, nonconserved coding sequence mutations (see, e.g., GrunderS., et al., Eur J Hum Genet. 2001, 9:672-676; Tokarz D., et al., Am JHum Genet. 2001; 69:629; each herein incorporated by reference in theirentireties).

Example 11

This example describes the identification and analysis of MR-1 genemutations in subjects with PDC. The MR-1 gene was identified as apositional candidate gene of unknown function and was expressed in thebrain. The National Center for Biotechnology Information database lists2 MR-1 transcripts (NM_(—)015488 and NM_(—)022572). These transcriptshave identical sequences for their last 8 exons but differ in theirfirst 2 exons. NM_(—)015488 is the larger transcript (3032 bp) andencodes a 385 amino acid protein with a predicted molecular weight of42.9 kDa. NM_(—)022572 is a slightly smaller transcript (2918 bp) thatencodes a 361 amino acid protein with a predicted molecular weight of40.7 kDa. Exons 1 and 2 (284 bp combined) of the larger transcriptcontain 47 bp of a 5_untranslated sequence and encode 79 amino acidsthat are not present in the smaller MR-1 transcript. Conversely, exon 1(165 bp) of the smaller transcript encodes 55 amino acids that areabsent in the larger MR-1 transcript (GenBank accession numbers:NM_(—)015488 and NM_(—)022572 for human MR-1 splice variants, AY299972for mouse MR-1, and NT_(—)005403 for the contig containing the MR-1gene). Each MR-1 exon (from NM_(—)015488 and NM_(—)022572) in affectedand unaffected subjects was sequenced from the PDC-Det and PDC-Pakindreds (see FIG. 9). Sequencing MR-1 NM_(—)015488 exon 1 in samplesfrom the PDCDet kindred revealed heterozygosity (both C and T) at MR-1NM_(—)015488 cDNA nucleotide 72 in each affected subject (n=8) (FIG. 9).With the exception of 2 previously identified nonpenetrant subjectsdiscussed as follows, each unaffected subject (n=17) and each controlsubject (n=105) had only C at this position, which agreed with the DNAsequence of contig NT_(—)005289 and the MR-1 gene's published cDNAsequence (NM_(—)015488). Sequencing MR-1 NM_(—)015488 exon 1 in samplesfrom the PDC-Pa kindred revealed heterozygosity (C and T) (FIG. 9) atMR-1 NM_(—)015488 cDNA nucleotide 66 (FIG. 9 and FIG. 10) in eachaffected subject (n=4). This mutation was absent in unaffected relatives(n=7) and control subjects (n=105). Sequencing each remaining MR-1NM_(—)015488 exon and each MR-1 NM_(—)022572 exon in 2 affected subjectsfrom the PDC-Det and PDC-Pa kindreds revealed no other mutation.

Previously, it was reported that unaffected subject PDCDet IV-2 (FIG. 9)contained the affected haplotype for PDC locus markers.7 The MR-1NM_(—)015488 exon 1 sequence analysis confirmed that this unaffectedindividual also had the C-to-T substitution at MR-1 nucleotide 72. Sheis thus a nonpenetrant individual as is her unaffected sibling (subjectPDC-Det IV-3) (FIG. 9), who along with her affected child has thesubstitution of T for C at MR-1 nucleotide 72.

The MR-1 NM_(—)015488 exon 1, the exon containing the C66T and C72Tmutations is found only in 1 MR-1 transcript variant of NM_(—)015488.Primers specific for this exon were created to examine MR-1 geneexpression by RT-PCR. These experiments detected the expression of MR-1NM_(—)015488 exon 1 only in the brain (FIG. 11). MR-1 NM_(—)015488 exon1 was not detected in the liver, kidney, skeletal muscle, heart, or lung(FIG. 11).

Mutations at C66T and C72T predict substitutions of valine for alanineat residue 9 (A9V) and residue 7 (A7V). The alanine residues atpositions 7 and 9 are part of an amino-terminal α helix that becomesdisrupted with either valine substitution (FIG. 12).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

1. A method for assessing a human subjects's risk for paroxysmaldystonic choreoathetosis comprising providing a sample from a humansubject, and detecting in said sample the presence of amyofibrillogenesis regulator 1 (MR-1) gene sequence variation, whereinsaid MR-1gene is SEQ ID NO:1, wherein said MR-1 gene sequence variationencodes a polymorphic MR-1 protein comprising SEQ ID NO:6 or SEQ ID NO:7or SEQ ID NO:8, wherein the presence of said MR-1 gene sequencevariation encoding said polymorphic MR-1 protein indicates an increasedrisk for paroxysmal dystonic choreoathetosis.
 2. The method of claim 1,wherein said detecting comprises detecting a polymorphic MR-1 protein.3. The method of claim 2, wherein said detecting a polymorphic MR-1protein occurs with an antibody.